KR102449653B1 - Kv1.3 potassium channel antagonists - Google Patents

Abstract

The present invention relates to compounds capable of selectively binding potassium channel Kv1.3 and inhibiting its activity. The present invention also relates to pharmaceutical compositions comprising such compounds and to the use of said compounds and said pharmaceutical compositions for the treatment or prophylaxis of autoimmune diseases, obesity, periodontitis and/or tissue transplant rejection.

Description

Kv1.3 potassium channel antagonist {KV1.3 POTASSIUM CHANNEL ANTAGONISTS}

The present invention relates to compounds capable of selectively binding potassium channel Kv1.3 and inhibiting its activity. The present invention also relates to pharmaceutical compositions comprising such compounds and to the use of said compounds and said pharmaceutical compositions for the treatment or prophylaxis of autoimmune diseases, obesity, periodontitis and/or tissue transplant rejection.

Autoimmune diseases are a group of more than 80 distinct diseases that occur when the host's immune response fails to distinguish between foreign antigens and self-molecules (autoantigens), thereby triggering an aberrant immune response. The cause of autoimmune diseases is still unknown, but it is thought to be caused by a combination of genetic and environmental factors.

Immune-inflammatory disorders such as multiple sclerosis, psoriasis and rheumatoid arthritis share a common pathogenic principle. Their pathogenesis is characterized by autoreactive memory T cells that mediate chronic inflammatory processes upon stimulation. In particular, repeated antigenic attacks, which usually occur in autoimmune diseases, induce long-lived central memory cells (T _CMs ), which, like naive cells, hominate to lymph nodes and come into contact with their cognate antigens, resulting in short-lived Differentiate into effector memory cells (T _EM ), which do not need to be homing to lymph nodes for antigen-induced activation. Activated T _EM cells turn into T _EM effectors and rapidly migrate to sites of inflammation, where they produce large amounts of pro-inflammatory cytokines. CD8 ⁺ T _EM cells are highly destructive as they additionally produce large amounts of perforin.

Current treatments for autoimmune diseases include systemic use of anti-inflammatory drugs and potent immunosuppressants and immunomodulators. However, these drugs cause numerous side effects, including, for example, suppressing the immune system as a whole, with the risk of infection and neoplasia. Moreover, in some patients, the drug cannot induce clinically significant remission.

The voltage dependent Kv1.3 K ⁺ channel is one of 76 potassium channels in the human genome and has been found to be present in human T lymphocytes. All human T cells express calcium-activated KCa3.1 as well as Kv1.3 channels, which together provide a balanced potassium excretion for calcium influx required for T cell activation and proliferation. The number of channels expressed by a given cell depends on its activation and differentiation state. Antigen or mitogen stimulated CD4 ⁺ and CD8 ⁺ T _EM cells display approximately 4- to 5-fold increased expression of Kv1.3, whereas human naive or T _CM cells up-regulated calcium-activated KCa3.1 channels. It regulates membrane potential and Ca2 ⁺ signaling in an activated state.

In view of differential overexpression in these T _EM cells, the Kv1.3 channel is associated with autoimmune diseases whose pathogenesis is associated with autoreactive T _EM cells, such as, for example, multiple sclerosis, rheumatoid arthritis, psoriasis and type 1 It constitutes a promising new T _EM -cell-specific therapeutic target for the treatment of diabetes as well as other chronic inflammatory diseases such as, for example, periodontitis.

Moreover, there are indications that the Kv1.3 channel plays a role in weight control. Thus, the Kv1.3 channel also constitutes a promising target for treating obesity.

Therefore, there is a continuing need for Kv1.3 channel-specific therapeutic compounds that exhibit strong and specific interactions with Kv1.3 channels and can block or decrease their activity.

One object of the present invention is to provide a compound capable of specifically binding to the potassium channel Kv1.3 and inhibiting or reducing its activity. A further object of the present invention is said inhibitory compound and said inhibitory compound for use in the treatment of autoimmune diseases such as for example multiple sclerosis, rheumatoid arthritis, psoriasis and/or type 1 diabetes mellitus, obesity and/or periodontitis To provide a pharmaceutical composition comprising

These and other objects as will be apparent from the following description and claims are obtained by the subject matter of the independent claims. Some of the preferred embodiments are defined by the dependent claims.

In a first aspect, the present invention relates to a compound comprising or consisting of the following amino acid sequence:

X ₁ -X ₂ -X ₃ -NVX ₄ -CX ₅ -X ₆ -X ₇ -X ₈ -X ₉ -CX ₁₀ -X ₁₁ -X ₁₂ -CX ₁₃ -X ₁₄ -X ₁₅ -TGCPX ₁₆ -X ₁₇ -KCMNRKCX ₁₈ -CX ₁₉ -X ₂₀ -C [SEQ ID NO: 23],

where X ₁ =T,Q,S,Y,N; X ₂ =I,F,V,A,L,W; X ₃ =I,T,Y,S,V,A,L; X ₄ =K,S,T,Y,R; X ₅ =R,T,K,S,Y; X ₆ =T,G,S,N,I,K,Q,A,V,L,Y; X ₇ =P,S,T; X ₈ =R,K,P; X ₉ =D,Q,N,E; X ₁₀ =A,Y,I,L,W,S,T,V,L,F; X ₁₁ =D,R,P,K,E,S,T,Y; X ₁₂ =P,H,V,I,L,A; X ₁₃ =R,K,Q,N; X ₁₄ =K,D,A,R,E,V,L,I; X ₁₅ =E,Q,A,L,D,N,V,I; X ₁₆ =Y,N,S,T,Q; X ₁₇ =A,G,V,I,L; X ₁₈ =K,R, X ₁₉ =Y,N,Q,T,S and X ₂₀ =G,R,K.

In a variant of the first aspect, the invention relates to a compound comprising or consisting of the amino acid sequence:

X ₁ -X ₂ -X ₃ -NVX ₄ -CX ₅ -X ₆ -X ₇ -X ₈ -X ₉ -CX ₁₀ -X ₁₁ -X ₁₂ -CX ₁₃ -X ₁₄ -X ₁₅ -TGCPX ₁₆ -X ₁₇ -KCMNRKCX ₁₈ -CX ₁₉ -X ₂₀ -C (SEQ ID NO: 23),

where X ₁ =T,Q,S,Y,N; X ₂ =I,F,V,A,L,W; X ₃ =I,T,Y,S,V,A,L; X ₄ =K,S,T,Y,R; X ₅ =R,T,K,S,Y; X ₆ =T,G,S,N,I,K,Q,A,V,L,Y; X ₇ =P,S,T; X ₈ =R,K,P; X ₉ =D,Q,N,E; X ₁₀ =A,Y,I,L,W,S,T,V,L,F; X ₁₁ =D,R,P,K,E,S,T,Y; X ₁₂ =P,H,V,I,L,A; X ₁₃ =R,K,Q,N; X ₁₄ =K,D,A,R,E,V,L,I; X ₁₅ =E,Q,A,L,D,N,V,I; X ₁₆ =Y,N,S,T,Q; X ₁₇ =A,G,V,I,L; X ₁₈ =K,R, X ₁₉ =Y,N,Q,T,S and X ₂₀ =G,R,K;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a second aspect, the present invention relates to a compound comprising or consisting of the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPX ₁₄ -X ₁₅ -KCMNRKCX ₁₆ -CX ₁₇ -X ₁₈ -C (SEQ ID NO: 1);

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,S,T,Q; X ₁₅ =A,G,V,I,L; X ₁₆ =K,R, X ₁₇ =Y,N,Q,T,S and X ₁₈ =G,R,K.

In a variant of the second aspect, the invention relates to a compound comprising or consisting of the amino acid sequence according to SEQ ID NO: 1,

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,S,T,Q; X ₁₅ =A,G,V,I,L; X ₁₆ =K,R, X ₁₇ =Y,N,Q,T,S and X ₁₈ =G,R,K;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a third aspect, the present invention relates to a compound comprising or consisting of the amino acid sequence:

GVX ₁ -INVX ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPX ₁₄ -X ₁₅ -KCMNRKCX ₁₆ -CX ₁₇ -X ₁₈ -C (SEQ ID NO: 27);

where X ₁ =P,I,F,V,A,L,W; X ₂ =K,S,T,Y,R; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,S,T,Q; X ₁₅ =A,G,V,I,L; X ₁₆ =K,R, X ₁₇ =Y,N,Q,T,S and X ₁₈ =G,R,K.

In a variant of the third aspect, the invention relates to a compound comprising or consisting of the amino acid sequence according to SEQ ID NO:27,

where X ₁ =P,I,F,V,A,L,W; X ₂ =K,S,T,Y,R; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,S,T,Q; X ₁₅ =A,G,V,I,L; X ₁₆ =K,R, X ₁₇ =Y,N,Q,T,S and X ₁₈ =G,R,K;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In another aspect the invention relates to a nucleic acid sequence encoding an amino acid sequence according to the invention.

In yet another aspect, the present invention further provides a vector comprising a nucleic acid sequence according to the present invention.

In yet another aspect, the present invention also provides a host cell comprising the nucleic acid sequence or vector according to the present invention.

In yet another aspect the invention relates to a pharmaceutical composition comprising a compound according to the invention.

The present invention in a further aspect also relates to a compound according to the invention or a pharmaceutical composition according to the invention for use in the treatment or prophylaxis of autoimmune diseases, obesity, periodontitis and/or tissue transplant rejection.

In another aspect the invention also relates to the use of a compound according to the invention or a pharmaceutical composition according to the invention for the manufacture of a medicament for the treatment or prevention of autoimmune diseases, obesity, periodontitis and/or tissue transplant rejection. is about

In another aspect the present invention relates to the treatment of an autoimmune disease, obesity, periodontitis and/or tissue transplant rejection in a mammal in need thereof, by administering to the mammal in need thereof a compound according to the present invention or a pharmaceutical composition according to the present invention. or how to prevent it.

The invention also in a further aspect provides a method for preparing a compound, nucleic acid sequence, vector or pharmaceutical composition according to the invention.

Figure 1 Measurement of Kv1.3, Kv1.5 and Kv1.1 channel inhibition by patch-clamps.
Figure 2 Sequence Listing cgtx 538-548.
Figure 3 Effect of compounds 1-8 (C1-8).
Compound 1: cgtx 538
Compound 2: cgtx 539
Compound 3: cgtx 540
Compound 4: cgtx 541
Compound 5: cgtx 542
Compound 6: cgtx 543
Compound 7: cgtx-544
Compound 8: cgtx 547
Left side illustrates hERG current traces after application of external solution (black), after compound application (blue) and after quinidine application (grey). The right panel shows the change in peak current amplitude over time. The dotted line indicates the compound application. The effect of each compound concentration was evaluated for 10 or 20 sweeps. All compounds were considered to have no or only small effect on hERG. Instead, quinidine completely blocked the current. It should be noted that a run down occurred from the start of compound application to the end of the recorded sweep.
Figure 4 Representative voltage-dependent activation of Kv1.3 channels expressed in Sf21 cells (a) and endogenously expressed in T _EM cells (b). (c) Pulse protocol for voltage-dependent Kv1.3 activation.
Figure 5 Characteristic normalized voltage-current-correlation of Kv1.3 channels expressed in Sf21 (middle and upper lines) and T _EM cells (lower line); n=41, n=12 and n=29, respectively. Using the Sf21 Baculovirus expression system, it is possible to express large amounts of Kv1.3 potassium channels.
Figure 6 Comparison of T _EM cells and Sf21 cells. Blocking of Kv1.3 channels expressed in Sf21 (a) and T _EM (b) cells by cgtx-544. Representative raw current traces and time dose responses in the presence of increasing concentrations of Kv1.3 antagonistic peptide are shown.
7 is a graphical representation of local induction of arthritis in the rat knee.
Fig . Swelling of the knee joint after induction of focal arthritis.
Figure 9 Immune status of laboratory animals based on WBC counts in peripheral blood; AI: Arthritis induction, naive animals = NaCl "immunized animals", immunized animals = animals treated with Freund's adjuvant with mBSA.
Figure 10 Knee joint between cgtx-544 treated and untreated control animals (each group: n=14; solid line with diamonds: untreated control animals; dashed line with squares: cgtx-544 treated animals) difference in swelling.
11 shows the difference in absolute knee swelling increase over time in arthritic rats using methotrexate (MTX) standard therapy. Antigen induced arthritis (AIA) was induced on day 0 by intra-articular injection of methylated BSA after two immunizations on days-21 and 14. The high-dose MTX regimen was administered at 1 mg/kg body weight of MTX starting on day-21 and 1×weekly sc until day 0. The low-dose MTX regimen was administered with 100 μg/kg body weight of MTX administered iv daily starting on day -3 until day 6, and the vehicle control group was administered 0.9% NaCl starting on day 0 and administered iv daily until day 6. The absolute increase in knee swelling is the difference between the non-induced and arthritis induced knee values, separately for each group. Values are mean ± SD. Dashed line with filled circles: MTX sc 1 x weekly group (group J, n=7); Long dashed line with triangle: MTX iv 1 x daily group (group L, n=7); Solid line with rhombuses: vehicle (NaCl) control (group H, n=14).
Figure 12. Difference in absolute knee swelling increase over time in arthritic rats with standard methotrexate (MTX) regimen compared to cgtx-544 peptide therapy over time. Antigen induced arthritis (AIA) was induced on day 0 by intra-articular injection of methylated BSA after two immunizations on days-21 and 14. The low-dose MTX regimen was administered iv daily starting on day -3 with 100 μg/kg body weight of MTX until day 6. In comparison, the cgtx-544 regimen group received cgtx-544 1 mg/kg body weight daily iv starting on day -3 and iv daily until day 6, while the vehicle control group also received 0.9% NaCl starting on day 0 daily iv administered. The absolute increase in knee swelling is the difference between the non-induced and arthritis induced knee values, separately for each group. Values are mean ± SD. Long dashed line with triangle: MTX iv 1x daily group (group L, n=7); dashed line with square: cgtx-544 therapy group (group I; n=14); Solid line with rhombuses: vehicle (NaCl) control (group H, n=14).
Figure 13. Differences in relative knee swelling increase over time in arthritic rats using methotrexate (MTX) standard therapy. Antigen induced arthritis (AIA) was induced on day 0 by intra-articular injection of methylated BSA after two immunizations on days-21 and 14. The high-dose MTX regimen was administered at 1 mg/kg body weight of MTX starting on day-21 and 1×weekly sc until day 0. The low-dose MTX regimen was administered with 100 μg/kg body weight of MTX administered iv daily starting on day -3 until day 6, and the vehicle control group was administered 0.9% NaCl starting on day 0 and administered iv daily until day 6. The relative increase in knee swelling is the difference between the non-induced and arthritic knee values, separately for each group after normalizing the values. Normalized values (Day-21=1) are mean±SD. Dashed line with filled circles: MTX sc 1 x weekly group (group J, n=7); Long dashed line with triangle: MTX iv 1 x daily group (group L, n=7); Solid line with rhombuses: vehicle (NaCl) control (group H, n=14).
Figure 14. Difference in relative knee swelling increase over time in arthritic rats with standard methotrexate (MTX) regimen compared to cgtx-544 regimen. Antigen induced arthritis (AIA) was induced on day 0 by intra-articular injection of methylated BSA after two immunizations on days-21 and 14. The low-dose MTX regimen was administered iv daily starting on day -3 with 100 μg/kg body weight of MTX until day 6. In comparison, the cgtx-544 regimen group received cgtx-544 1 mg/kg body weight daily iv starting on day -3 and iv daily until day 6, while the vehicle control group also received 0.9% NaCl starting on day 0 daily iv administered. The relative increase in knee swelling is the difference between the non-induced and arthritic knee values, separately for each group after normalizing the values. Normalized values (Day-21=1) are mean±SD. Long dashed line with triangle: MTX iv 1x daily group (group L, n=7); dashed line with square: cgtx-544 peptide therapy group (group I; n=14); Solid line with rhombuses: vehicle (NaCl) control (group H, n=14).
Figure 15 Immune status of arthritic rats with methotrexate (MTX) standard therapy as compared to cgtx-544 therapy. absolute white blood cell (WBC) count; Values are mean ± SD (n=7). Dashed line with filled circles: MTX sc 1 x weekly group (group J, n=7); Long dashed line with triangle: MTX iv 1 x daily group (group L, n=7); dashed line with square: cgtx-544 peptide therapy iv 1 x daily group (group I, n=7); Solid line with rhombic: vehicle (NaCl) control iv 1 x daily group (group H, n=7). Black arrow: M. Immunization time with M. tuberculosis (750 μg) and mBSA. EDTA leukocytes were measured using Sysmex XT-2000i.V.
Figure 16. Neutrophil granulocytes over time in arthritic rats using methotrexate (MTX) standard therapy as compared to cgtx-544 therapy. a. Absolute values are mean ± SD (n=7). Dashed line with filled circles: MTX sc 1 x weekly group (group J, n=7); Long dashed line with triangle: MTX iv 1 x daily group (group L, n=7); Dashed line with square: cgtx-544 regimen iv 1 x daily group (Group I, n=7); Solid line with rhombic: vehicle (NaCl) control iv 1 x daily group (group H, n=7). Black arrow: M. Immunization times with tuberculosis (750 μg) and mBSA. b. Relative neutrophils (%) are related to the total amount of WBC. Values are mean ± SD (n=7). Dashed line with filled circles: MTX sc 1 x weekly group (group J, n=7); Long dashed line with triangle: MTX iv 1 x daily group (group L, n=7); Dashed line with square: cgtx-544 regimen iv 1 x daily group (Group I, n=7); Solid line with rhombic: vehicle (NaCl) control iv 1 x daily group (group H, n=7). Black arrows (estimated days-21 and 14): M. Immunization times with tuberculosis (750 μg) and mBSA. EDTA leukocytes were measured using Sysmex XT-2000i.V.
Figure 17 Lymphocytes over time in arthritic rats with methotrexate (MTX) standard therapy as compared to cgtx-544 therapy. a. Absolute values are mean ± SD (n=7). Dashed line with filled circles: MTX sc 1 x weekly group (group J, n=7); Long dashed line with triangle: MTX iv 1 x daily group (group L, n=7); Dashed line with square: cgtx-544-peptide therapy iv 1 x daily group (Group I, n=7); Solid line with rhombic: vehicle (NaCl) control iv 1 x daily group (group H, n=7). Black arrow: M. Immunization times with tuberculosis (750 μg) and mBSA. b. Percentage of Lymphocytes in Arthritic Rats with Standard of Methotrexate (MTX) Therapy compared to cgtx-544 Therapy—Relative Lymphocytes (%) correlates with the total amount of WBC. Values are mean ± SD (n=7). Dashed line with filled circles: MTX sc 1 x weekly group (group J, n=7); Long dashed line with triangle: MTX iv 1 x daily group (group L, n=7); Dashed line with square: cgtx-544 regimen iv 1 x daily group (Group I, n=7); Solid line with rhombic: vehicle (NaCl) control iv 1 x daily group (group H, n=7). Black arrow: M. Immunization times with tuberculosis (750 μg) and mBSA. EDTA leukocytes were measured using Sysmex XT-2000i.V.
Figure 18 Prophylactic treatment and dose dependence—relative increase in knee diameter over time (high/medium/low dose). Values are mean ± SD. Long dashed line with triangle: cgtx-544 peptide 5 mg/kg body weight therapy group (group N, n=5); dashed line with black square: cgtx-544 peptide 1 mg/kg body weight therapy group (group I, n=14); Dashed line with solid circles: cgtx-544 peptide 0.1 mg/kg body weight therapy group (group M, n=5); Solid line with rhombuses: vehicle (NaCl) control (group H #15-19, n=5). Solid line with white square: no uninduced knee diameter increase (groups I, N, M and H; uninduced left knee).
Figure 19 Statistical analysis of cgtx-544 efficacy results: with and without cgtx-544 peptide therapy (high/medium/low dose) on days 1 (a) and 3 (b) after arthritis induction Differences in relative knee swelling. All box plots present the median, interquartile range, sample minimum and maximum. For significance analysis, the vehicle control group was compared to the therapy group. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 20 Difference in knee diameter in arthritic rats with and without curative treatment - treatment started at d=0 or d=1 - cgtx-544 peptide therapy (treatment started at d0 and d1) (In-individual, induced knee diameter minus non-derived knee diameter) is plotted over time. Values are mean ± SD. Long dashed line with triangle - start treatment on d1: cgtx-544 peptide 1 mg/kg body weight therapy group (group Q, n=7); dotted line with solid circle - treatment started on d0: cgtx-544 peptide 1 mg/kg body weight therapy group (group P, n=6); Solid line with rhombuses: vehicle (NaCl) control (group H). Solid line with white square: no uninduced knee diameter increase (groups Q, P and H; uninduced left knee).
Figure 21 Statistical analysis of cgtx-544 efficacy results on days 1 (a), 2 (b) and 3 (c) after arthritis induction: cgtx-544 peptide therapy - curative treatment (start at d0/d1) The difference in relative knee swelling with and without). All box plots present the median, interquartile range, sample minimum and maximum. For significance analysis, the vehicle control group was compared to the therapy group. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 22. Time WBC counts in arthritic rats with and without curative treatment - treatment started at d=0 or d=1 - cgtx-544 peptide therapy (treatment started at d0 and d1) It is shown according to the process. Absolute white blood cell (WBC) count: Values are mean±SD. Long dashed line with triangle - start treatment on d1: cgtx-544(Mix) peptide 1 mg/kg body weight therapy group (group Q, n=7); dotted line with solid circle - treatment started on d0: cgtx-544 peptide 1 mg/kg body weight therapy group (group P, n=6); Solid line with rhombic: vehicle (NaCl) control (group H, n=5). Black arrow: M. Immunization times with tuberculosis (750 μg) and mBSA.
23. Percentage of WBC in Arthritic Rats with and Without Curative Treatment —Treatment Started at d=0 or d=1—Cgtx-544 Peptide Therapy (Treatment Started at d0 and D1) Relative amounts of neutrophil granulocytes are plotted over time. Neutrophils (%) relative to the total amount of WBC. Values are mean ± SD. Long dashed line with triangle - start treatment on d1: cgtx-544 peptide 1 mg/kg body weight therapy group (group Q, n=7); dotted line with solid circle - treatment started on d0: cgtx-544 peptide 1 mg/kg body weight therapy group (group P, n=6); Solid line with rhombic: vehicle (NaCl) control (group H, n=5). Black arrow: M. Immunization times with tuberculosis (750 μg) and mBSA.
Figure 24. Percentage of WBCs in Arthritic Rats with and Without Curative Treatment—Treatment Started at d=0 or d=1—Cgtx-544 Peptide Therapy (Treatment Started at d0 and D1) The relative amount of lymphocytes is plotted over time. Lymphocytes (%) relative to total amount of WBC. Values are mean ± SD. Long dashed line with triangle - start treatment on d1: cgtx-544 peptide 1 mg/kg body weight therapy group (group Q, n=7); dotted line with solid circle - treatment started on d0: cgtx-544 peptide 1 mg/kg body weight therapy group (group P, n=6); Solid line with rhombic: vehicle (NaCl) control (group H, n=5). Black arrow: M. Immunization times with tuberculosis (750 μg) and mBSA.
Figure 25 Therapeutic treatment (once a week or twice a week)—relative increase in knee joint diameter over time. Values are mean ± SD. Long dashed line with triangle - single treatment on d1: cgtx-544 peptide 1 mg/kg body weight therapy group (group R6-10, n=5); dashed line with solid circles—single treatment on d1 and d4: cgtx-544 peptide 1 mg/kg body weight therapy group (group R1-5, n=5); Both groups were combined until day 4. Solid line with rhombic: vehicle (NaCl) control (group H, n=5).
Figure 26 Therapeutic treatment (once a week or twice a week) - Statistical analysis of cgtx-544 efficacy: with cgtx-544 peptide therapy on days 2 (A) and 5 (B) after arthritis induction and the difference in relative knee swelling when not used. All box plots present the median, interquartile range, sample minimum and maximum. For significance analysis, the vehicle control group was compared to the therapy group. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 27 Analysis of mBSA specific antibodies. In experimental groups H and I, all animals produced antibodies against the antigen mBSA during the period of immunization and AIA induction. No specific mBSA antibody could be detected in control F (naive animals).
Figure 28 Statistical analysis of MTX efficacy results: difference in relative knee swelling with and without MTX therapy (high/low dose) on days 1 (A) and 3 (B) after arthritis induction. All box plots present the median, interquartile range, sample minimum and maximum. For significance analysis, the vehicle control group was compared to the therapy group. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant.
29 Schematic of peptide synthesis.
Figure 30 UPLC profile of cgtx-544 peptide after folding.
Figure 31 IC50 of cgtx-544 (Sing). a. cgtx-544 (Sing) displayed an IC50 value of 6.9 nM when fitting the results to a Hill curve. b. Step-by-step current reduction with increasing concentrations of cgtx-544 (Sing). c. Current traces during measurements at different concentrations of cgtx-544 (Sing).
Figure 32 Selectivity of cgtx-544 (Sing). a. cgtx-544(Sing) displayed an IC50 value of about 6 μM on Kv1.1 when fitting the results to the Hill curve. b. Step-by-step current reduction with increasing concentrations of cgtx-544 (Sing) on Kv1.1. c. Current traces during measurements at different concentrations of cgtx-544 (Sing) on Kv1.1. d. A 10 μM solution of cgtx-544(Sing) did not induce a significant decrease in the Kv1.5 current, whereas the current was sensitive to quinidine. e. A 100 nM solution of cgtx-544(Sing) did not alter the Kv1.2 current in stably transfected CHO cells. Concentrations >1 μM only resulted in slightly reduced current. IC50 was established at 2.5 μM.
Figure 33. Disintegration of cgtx-544 (Sing) in human serum at 37°C. A known amount of cgtx-544 (Sing) was added to human blood serum from three blood donors and incubated at 37° C. for a period of 57 days. The blocking activity of the peptides was measured on the Kv1.3 channel over this period. The peptide was still stable for 16 hours and could still be detected after 45 days. At 57 days, the blocking effect was no longer visible. The decay is depicted as a decrease in the concentration of cgtx-544 (Sing), as determined as C(t) = IC50 (t ₀ )/IC50(t).C ₀ . This decay is a simple decay curve: C(t) = C 0.2(-t/t ^1/2 ), where C ₀ is the initial concentration of the peptide in solution, t ₀ is a ₀ min incubation at the 37°C point and , t _1/2 is the half-life).
Figure 34 Reduction of cgtx-544 (Sing) in the serum of treated rats after iv injection. The concentration of unbound circulating cgtx-544 (Sing) was calculated based on a standard curve. The standard error of the mean is plotted as error bars (n=6).
Figure 35 A. Prolonged incubation of cgtx-544 produces picomolar IC50 values. Kv1.3 currents were normalized to initial peak currents and complete blockade by the non-specific potassium channel blocker quinidine (data not shown). Under control conditions (upper curve, n=11), the current is stable for at least 10 min, but a single application of cgtx-544 (lower curve, n=13) (arrow) induces blockage, which increases with time do. B. Dose response curves of cgtx-544 at extended incubation times. In the dashed black line, the dose response curve for cgtx-544 using short incubation times is presented. The solid black curve is the result of incubating cgtx-544 with Kv1.3 channels for an extended period of time, i.e. 15 min. Due to the 20% blockade observed with the short incubation time (white dot) compared to the 60% block with the long incubation time (black dots), the dose response curve shifted to the left, and thus the IC50 (horizontal dashed line) ) is lower. C. IC50 values of cgtx-544 on Kv1.3 and Kv1.1 with long incubation times and short incubation times.

Before the present invention is described in detail below, it is to be understood that the present invention is not limited to the particular methodologies, protocols, and reagents described herein, as they may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and does not limit the scope of the invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. As used herein, one-letter and three-letter amino acid abbreviations correspond to IUPAC nomenclature (see, eg, European Journal of Biochemistry, 138:9-37, 1984).

The following definitions are introduced:

As used in this specification and the intended claims, the singular forms also include the respective plural unless the context clearly dictates otherwise.

It is to be understood that the term “comprises” and variations such as “comprising” are not limiting. For the purposes of the present invention, the term “consisting of” is considered to be a preferred embodiment of the term “comprising”. If later a particular group is defined to include at least a certain number of embodiments, this is also meant to encompass the group which preferably consists only of these embodiments.

The terms “about” and “approximately” in the context of the present invention mean an accuracy interval that the person skilled in the art will understand will still ensure the technical effect of the feature in question. The term typically encompasses a deviation of ±10% and preferably ±5% from the indicated numerical value.

As used herein, the term “peptide” refers to a molecular chain of amino acids and does not refer to a product of a specific length; Polypeptides, oligopeptides and proteins are included within the definition of peptides. Peptides according to this definition may be both naturally occurring peptides and synthetic peptides which may include naturally occurring or non-naturally occurring amino acids. Within this definition are functional derivatives of said peptides, i.e. by modifying the side chain, free amino and/or carboxy-terminus of naturally occurring or non-naturally occurring amino acids, for example, preferably without changing the identity of each amino acid, Chemically modified peptides are further included. For example, the side chain or free amino or carboxy terminus of an amino acid of a particular peptide may be modified, for example, by glycosylation, amidation, phosphorylation, ubiquitination, carboxylation, and the like. In a preferred embodiment the peptides according to the invention may be modified by PEGylation, HESylation or PASylation.

The compounds disclosed herein are surprisingly, Kv1 compared to other potassium channels such as for example Kv1.1, Kv1.2, Kv1.5, Kv1.6, IKCa1, hERG or high conductance Ca2+-activated K+ channels (BK channels). .3 It has been found by the inventors of the present invention that it can selectively bind potassium channels. Given the fact that Kv1.3 is predominant in T _EM cells, the compounds according to the invention thus constitute a potent therapeutic agent for T _EM -cell mediated diseases, such as for example autoimmune diseases. Moreover, the compounds disclosed herein provide the benefit of reduced side effects, since they do not substantially modulate the activity of other potassium channels distributed in other types of cells or tissues.

Accordingly, the present invention generally relates to compounds capable of selectively binding Kv1.3 potassium channels over other potassium channels, such as, for example, Kv1.1. Such compounds are contemplated for use in the treatment or prevention of autoimmune diseases. The compound may be selected from the group consisting of peptides, antibodies or small molecules. In a particularly preferred embodiment said compounds are peptides and variants thereof as described for the first, second and third aspects of the invention.

The antibody capable of selectively binding potassium channel Kv1.3 may be a monoclonal or polyclonal antibody. In some embodiments the antibody is also an antibody variant or fragment, such as for example a single chain antibody, diabody, minibody, single chain Fv fragment (sc(Fv)), sc(Fv) ₂ antibody, Fab fragment or F (ab') ₂ fragments, provided that the antibody variant or fragment is capable of selectively binding potassium channel Kv1.3.

As used herein, the term “small molecule” refers to a small organic compound having a low molecular weight.

A small molecule can be a synthetic compound not known to occur in nature, or a naturally occurring compound isolated from a natural source, such as, for example, a cell, plant, fungus, animal, or the like, or known to occur in a natural source. Small molecules in the context of the present invention preferably have a molecular weight of less than 5000 daltons, more preferably less than 4000 daltons, more preferably less than 3000 daltons, more preferably less than 2000 daltons or even more preferably less than 1000 daltons. In a particularly preferred embodiment small molecules in the context of the present invention have a molecular weight of less than 800 daltons.

In another preferred embodiment small molecules in the context of the present invention have a molecular weight of 50 to 3000 Daltons, preferably 100 to 2000 Daltons, more preferably 100 to 1500 Daltons and even more preferably 100 to 1000 Daltons. Most preferably small molecules in the context of the present invention have a molecular weight of between 100 and 800 daltons.

Small molecules capable of selectively binding the potassium channel Kv1.3 can be identified, for example, by screening a small library of compounds.

Accordingly, the present invention in the first, second and third aspects and variants thereof relates to peptide compounds capable of selectively binding potassium channel Kv1.3. Preferably, the compound is a potassium channel compared to other potassium channels, such as for example Kv1.1, Kv1.2, Kv1.5, Kv1.6, IKCa1, hERG or high conductance Ca2+-activated K+ channels (BK channels). It can bind selectively to Kv1.3. In a particularly preferred embodiment, the compounds according to the invention are capable of selectively binding potassium channel Kv1.3 compared to potassium channel Kv1.1.

In a first aspect, the compound according to the invention comprises or consists of the following amino acid sequence:

X ₁ -X ₂ -X ₃ -NVX ₄ -CX ₅ -X ₆ -X ₇ -X ₈ -X ₉ -CX ₁₀ -X ₁₁ -X ₁₂ -CX ₁₃ -X ₁₄ -X ₁₅ -TGCPX ₁₆ -X ₁₇ -KCMNRKCX ₁₈ -CX ₁₉ -X ₂₀ -C (SEQ ID NO: 23),

where X ₁ =T,Q,S,Y,N; X ₂ =I,F,V,A,L,W; X ₃ =I,T,Y,S,V,A,L; X ₄ =K,S,T,Y,R; X ₅ =R,T,K,S,Y; X ₆ =T,G,S,N,I,K,Q,A,V,L,Y; X ₇ =P,S,T; X ₈ =R,K,P; X ₉ =D,Q,N,E; X ₁₀ =A,Y,I,L,W,S,T,V,L,F; X ₁₁ =D,R,P,K,E,S,T,Y; X ₁₂ =P,H,V,I,L,A; X ₁₃ =R,K,Q,N; X ₁₄ =K,D,A,R,E,V,L,I; X ₁₅ =E,Q,A,L,D,N,V,I; X ₁₆ =Y,N,S,T,Q; X ₁₇ =A,G,V,I,L; X ₁₈ =K,R, X ₁₉ =Y,N,Q,T,S and X ₂₀ =G,R,K.

In a variant of the first aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO:23:

where X ₁ =T,Q,S,Y,N; X ₂ =I,F,V,A,L,W; X ₃ =I,T,Y,S,V,A,L; X ₄ =K,S,T,Y,R; X ₅ =R,T,K,S,Y; X ₆ =T,G,S,N,I,K,Q,A,V,L,Y; X ₇ =P,S,T; X ₈ =R,K,P; X ₉ =D,Q,N,E; X ₁₀ =A,Y,I,L,W,S,T,V,L,F; X ₁₁ =D,R,P,K,E,S,T,Y; X ₁₂ =P,H,V,I,L,A; X ₁₃ =R,K,Q,N; X ₁₄ =K,D,A,R,E,V,L,I; X ₁₅ =E,Q,A,L,D,N,V,I; X ₁₆ =Y,N,S,T,Q; X ₁₇ =A,G,V,I,L; X ₁₈ =K,R, X ₁₉ =Y,N,Q,T,S and X ₂₀ =G,R,K;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a preferred embodiment of the first aspect, the compound according to the invention comprises or consists of the following amino acid sequence:

X ₁ -X ₂ -X ₃ -NVX ₄ -CX ₅ -X ₆ -X ₇ -X ₈ -X ₉ -CX ₁₀ -X ₁₁ -X ₁₂ -CX ₁₃ -X ₁₄ -X ₁₅ -TGCPX ₁₆ -X ₁₇ -KCMNRKCX ₁₈ -CX ₁₉ -X ₂₀ -C (SEQ ID NO: 24);

where X ₁ =T,Q; X ₂ =I,F; X ₃ =I,T; X ₄ =K,S; X ₅ =R,T,K; X ₆ =T,G,S,N,I; X ₇ =P,S; X ₈ =R,K,P; X ₉ =D,Q,N,E; X ₁₀ =A,Y,I,L,W; X ₁₁ =D,R,P,K,E,S; X ₁₂ =P,H,V; X ₁₃ =R,K,Q; X ₁₄ =K,D,A,R; X ₁₅ =E,Q,A,L; X ₁₆ =Y,N; X ₁₇ =A,G; X ₁₈ =K,R, X ₁₉ =Y,N and X ₂₀ =G,R.

In a preferred embodiment of a variant of the first aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO: 24:

where X ₁ =T,Q; X ₂ =I,F; X ₃ =I,T; X ₄ =K,S; X ₅ =R,T,K; X ₆ =T,G,S,N,I; X ₇ =P,S; X ₈ =R,K,P; X ₉ =D,Q,N,E; X ₁₀ =A,Y,I,L,W; X ₁₁ =D,R,P,K,E,S; X ₁₂ =P,H,V; X ₁₃ =R,K,Q; X ₁₄ =K,D,A,R; X ₁₅ =E,Q,A,L; X ₁₆ =Y,N; X ₁₇ =A,G; X ₁₈ =K,R, X ₁₉ =Y,N and X ₂₀ =G,R;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a more preferred embodiment of the first aspect and variants thereof, the compound according to the invention comprises or consists of the amino acid sequence of SEQ ID NO: 25 (cgtx-544) or SEQ ID NO: 26 (cgtx 547). In this context, in another more preferred embodiment of the first aspect and variants thereof, the compound according to the invention has at least about 50%, at least about 60%, at least about 70% with SEQ ID NO: 25 (cgtx-544) , an amino acid sequence having a percent identity of at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. Determination of percent identity between two sequences is preferably carried out as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci USA 90: 5873-5877]. Such algorithms are described, for example, in Altschul et al. available at NCBI ( http://www.ncbi.nlm.nih.gov/blast/Blast.cge ). (1990) J. Mol. Biol. 215: 403-410]. Determination of percent identity is preferably performed using standard parameters of the BLASTn and BLASTp programs. BLAST polynucleotide searches are preferably performed using the BLASTn program. For general parameters, the "Maximum Target Sequence" box can be set to 100, the "Short Lookup" box can be checked, the "Expected Threshold" box can be set to 10 and the "Word Size" box can be set to 28 can be set. For the scoring parameters, "Match/Mismatch Score" can be set to 1, -2, and the "Gap Cost" box can be set to linear. For filter and masking parameters, you can uncheck the "Low Complexity Regions" box, uncheck the "Species Specific Repeat Sequences" box, and check the "Masks for Lookup Tables Only" box. You can, or you can uncheck the "Mask lowercase" box. BLAST protein searches are preferably performed using the BLASTp program. For general parameters, the "Maximum Target Sequence" box can be set to 100, the "Short Lookup" box can be checked, the "Expected Threshold" box can be set to 10 and the "Word Size" box can be set to "3"" can be set. For the scoring parameters, the "Matrix" box can be set to "BLOSUM62", the "Gap Cost" box can be set to "Existence: 11 Extension:1", the "Configuration Adjustment" box can be set to "Conditional Composition Score Matrix Adjustment" can be set to For filter and masking parameters, you may uncheck the "Low Complexity Region" box, you may uncheck the "Mask for lookup tables only" box, and you may uncheck the "Mask lowercase" box .

In an even more preferred embodiment of the first aspect and variants thereof, the compound according to the invention comprises or consists of the amino acid sequence of SEQ ID NO: 25 (cgtx-544).

The embodiments described below preferably refer to the compounds of the first aspect of the invention and variants thereof. However, these embodiments, e.g., the length of the peptide as discussed below, the selectivity for Kv1.3 compared to Kv1.1, etc., are not limited to the second and third aspects of the invention and modifications thereof, as well as modifications thereof, as described below. It is necessary to understand that the same applies to the compounds of their preferred embodiments.

In a further preferred embodiment the compounds according to the invention are peptides.

When the compound according to the invention is a peptide, the compound according to the invention is preferably less than 1000 amino acids, less than 500 amino acids, less than 200 amino acids, preferably less than 150 amino acids, less than 100 amino acids. , less than 90 amino acids, less than 80 amino acids, less than 70 amino acids, less than 60 amino acids, less than 50 amino acids, or less than 40 amino acids.

In a further preferred embodiment the compounds according to the invention contain at least 20 to 1000 amino acids, preferably at least 25 to 500 amino acids, more preferably at least 30 to 200 amino acids, more preferably at least 34 to 150 amino acids. amino acids, more preferably at least 34 to 100 amino acids, more preferably at least 34 to 90 amino acids, and even more preferably at least 34 to 80 amino acids, at least 34 to 70 amino acids, 34 to 60 amino acids amino acids, 34 to 50 amino acids or 34 to 40 amino acids in length.

In a more preferred embodiment the compounds according to the invention consist essentially of the amino acid sequence as indicated.

The amino acids comprised in the compounds according to the invention may be L- or D-amino acids. Preferably, the amino acids comprised in the compounds according to the invention are L-amino acids. In some embodiments, D-amino acids may be preferred, particularly where high resistance to proteolysis is desired.

Compounds that simultaneously inhibit the activity of different potassium channels may exhibit significant side effects when applied to living organisms because the different potassium channels are differentially distributed in different types of cells. Accordingly, the present invention provides compounds capable of selectively binding potassium channel Kv1.3 when compared to other potassium channels, in particular potassium channel Kv1.1.

The term "selectively binds" as used herein means that the compound according to the invention is compared to other ion channels, in particular such as for example Kv1.1, Kv1.2, Kv1.5, Kv1.6, IKCa1, preferential binding to Kv1.3 channels compared to other potassium channels such as hERG or large conductance Ca2+-activated K+ channels (BK channels).

Preferably, the compound according to the invention is capable of selectively binding potassium channel Kv1.3 compared to potassium channel Kv1.1. In this case, the compound according to the invention binds to the Kv1.3 channel with a substantially higher affinity than when it binds to the Kv1.1 channel.

The toxin HsTx 1 in the context of the present invention is not considered as a compound capable of selectively binding the potassium channel Kv1.3, since the toxin binds and strongly inhibits the Kv1.1 channel.

In a preferred embodiment according to the invention a compound that selectively binds to Kv1.3 is at least 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold compared to the K _d value when such a compound binds to a Kv1.1 channel. binds the potassium channel Kv1.3 with a K _d value that is fold, 2000 fold, preferably at least 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 fold lower. In a further preferred embodiment according to the invention a compound that selectively binds Kv1.3 is such that the compound binds to other potassium channels, such as for example Kv1.1, Kv1.2, Kv1.5, Kv1.6, IKCa1, hERG. or binds the potassium channel Kv1.3 with a K _d value at least 4000, 5000 or 10000 fold lower when compared to the K _d value when binding to a high conductivity Ca2+-activated K+ channel (BK channel).

In a particularly preferred embodiment the compounds which selectively bind to Kv1.3 according to the invention have a K _d value at least 4000, 5000 or 10000 times lower compared to the K _d value when such a compound binds to a Kv1.1 channel. Binds with potassium channel Kv1.3.

In a preferred embodiment, the compounds according to the invention are typically from about 0.1 nM to about 250 nM, from about 0.5 nM to about 250 nM, from about 1 nM to about 225 nM, from about 1 nM to about 200 nM, even more preferably about It binds the potassium channel Kv1.3 with a K _d value of from 1 nM to about 100 nM, and most preferably from about 1 nM to about 50 nM.

The compounds according to the invention preferably have a K _d value of greater than 0.1 mM, greater than 0.2 mM, greater than 0.3 mM, greater than 0.4 mM or greater than 0.5 mM other potassium channels, such as for example Kv1.1, Kv1.2, Kv1. 5, binds to Kv1.6, IKCa1, hERG or large conductance Ca2+-activated K+ channels (BK channels). In a particularly preferred embodiment the compounds according to the invention bind Kv1.1 channels with a K _d value of greater than 0.1 mM, greater than 0.2 mM, greater than 0.3 mM, greater than 0.4 mM or greater than 0.5 mM.

Accordingly, the compounds according to the invention preferably bind the potassium channel Kv1.3 with a K _d value of from about 0.5 to about 200 nM, such as from about 1 or 2 nM to about 200 nM, greater than 0.1 mM, greater than 0.2 mM, Binds to Kv1.1 channels with K _d values greater than 0.3 mM, greater than 0.4 mM or greater than 0.5 mM. More preferably, the compound according to the invention binds the potassium channel Kv1.3 with a K _d value of from about 0.5 to about 100 nM, such as from about 1 or 2 nM to about 100 nM, and is greater than 0.1 mM, greater than 0.2 mM, 0.3 Binds to Kv1.1 channels with K _d values greater than mM, greater than 0.4 mM or greater than 0.5 mM. Even more preferably, the compound according to the invention binds the potassium channel Kv1.3 with a K _d value of about 0.5 to about 50 nM, such as about 1 or 2 nM to about 50 nM, and with a K _d value of greater than 0.5 mM. Binds to the Kv1.1 channel.

The compound according to the invention is preferably capable of selectively binding potassium channel Kv1.3 and blocking or reducing its activity. The compound according to the present invention has a potassium channel Kv1.3 activity of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, more preferably 80% or more compared to the control. % or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In some preferred embodiments the compounds according to the invention are capable of blocking the activity of the potassium channel Kv1.3 by 100%.

The ability of a compound according to the invention to selectively bind to a Kv1.3 channel and to block or decrease its activity can be tested by assays known in the art. For example, in one approach Kv1.3 or another potassium channel, such as for example Kv1.1, Kv1.2, Kv1.5, Kv1.6, IKCa1, hERG or high conductance Ca2+-activated K+ channel Mammalian cell lines expressing (BK channels) can be contacted with the compounds of the invention and channel currents can be measured, for example, as described in Grissmer et al. (Mol Pharmacol 45; 1227-34 (1994))] or by the patch clamp method as described in the Examples section herein below. Each compound can be tested, for example, at different concentrations. The K _d values of the compounds according to the invention can then be determined by fitting the Hill equation to the measured reduction in peak current.

For example, the method of determining K _d as described in Example 2 does not take into account the number of channels being measured, and for the purposes of this disclosure it should be understood why K _d corresponds to the IC50 value (term K _d and IC 50 are used synonymously herein). It should be understood that lower K _d values may be observed when using fully folded and purified peptides compared to mixtures comprising incompletely folded peptides (see Example 2).

In some embodiments a compound according to the invention is capable of attaching to its N-terminal amino group or its C-terminal carboxy group an antibody or other molecule capable of recognizing and targeting T _EM cells.

The compounds of the present invention can be prepared using techniques known in the art. For example, peptides are initially described, for example, in Merrifield, J. Am. Chem. Soc. 85; 7129 (1963) and later in Meienhofer et al., J. Peptide Prot. Res. 13; 35 (1979) and Fields, et al., Peptide Res. 4; 95 (1991)], and can be synthesized using solid phase Fmoc chemistry. Such synthesis can be performed, for example, on an automated peptide synthesizer. Once synthesized, the sequence can be verified using an automated peptide sequencer.

Potassium channels mentioned in the context of the present invention, such as for example potassium channels Kv1.1, Kv1.2, Kv1.3, Kv1.5, Kv1.6, IKCa1, hERG or large conductance Ca2+-activated K+ channels (BK channels) are well known in the art. Thus, the skilled artisan will know the polynucleotide and amino acid sequences of these channels from any suitable public database, such as, for example, the NCBI database (eg http://www.ncbi.nlm.nih.gov/pubmed/), and Its orthologs and splice isoforms can be easily retrieved.

In a second aspect, the present invention relates to a compound comprising or consisting of the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPX ₁₄ -X ₁₅ -KCMNRKCX ₁₆ -CX ₁₇ -X ₁₈ -C (SEQ ID NO: 1);

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,S,T,Q; X ₁₅ =A,G,V,I,L; X ₁₆ =K,R, X ₁₇ =Y,N,Q,T,S and X ₁₈ =G,R,K.

In a preferred embodiment, the compound is not HsTx 1 (SEQ ID NO: 32).

In a variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO:1:

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,S,T,Q; X ₁₅ =A,G,V,I,L; X ₁₆ =K,R, X ₁₇ =Y,N,Q,T,S and X ₁₈ =G,R,K;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a preferred embodiment, said compound is not HsTx 1.

In a preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPX ₁₄ -X ₁₅ -KCMNRKCX ₁₆ -CX ₁₇ -X ₁₈ -C (SEQ ID NO: 2);

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =Y,N; X ₁₅ =A,G; X ₁₆ =K,R, X ₁₇ =Y,N and X ₁₈ =G,R;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a preferred embodiment, said compound is not HsTx 1.

In a preferred embodiment of a variant of the second aspect, the invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO: 2:

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =Y,N; X ₁₅ =A,G; X ₁₆ =K,R, X ₁₇ =Y,N and X ₁₈ =G,R;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a preferred embodiment, said compound is not HsTx 1.

In a preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPNAKCMNRKCX ₁₄ -CX ₁₅ -X ₁₆ -C ( SEQ ID NO: 3);

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =K,R, X ₁₅ =Y,N,Q,T,S and X ₁₆ =G,R,K.

In a preferred embodiment of a variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO:3:

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =K,R, X ₁₅ =Y,N,Q,T,S and X ₁₆ =G,R,K;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a more preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPNAKCMNRKCX ₁₄ -CX ₁₅ -X ₁₆ -C ( SEQ ID NO: 4);

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =K,R, X ₁₅ =Y,N and X ₁₆ =G,R.

In a more preferred embodiment of the variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO:4:

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =K,R, X ₁₅ =Y,N and X ₁₆ =G,R;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In another preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPX ₁₄ -X ₁₅ -KCMNRKCX ₁₆ -CYGC ( SEQ ID NO: 5);

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,S,T,Q; X ₁₅ =A,G,V,I,L and _X16 =K,R.

In another preferred embodiment of the variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO:5:

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,S,T,Q; X ₁₅ =A,G,V,I,L and _X16 =K,R;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a more preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPX ₁₄ -X ₁₅ -KCMNRKCX ₁₆ -CYGC ( SEQ ID NO: 6);

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =Y,N; X ₁₅ =A,G and X ₁₆ =K,R.

In a more preferred embodiment of a variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO:6:

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =Y,N; X ₁₅ =A,G and X ₁₆ =K,R;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In another preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPNAKCMNRKCX ₁₄ -CYGC (SEQ ID NO: 7) ;

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I and X ₁₄ =K,R.

In another preferred embodiment of the variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO:7:

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I and X ₁₄ =K,R;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a more preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPNAKCMNRKCX ₁₄ -CYGC (SEQ ID NO: 8) ;

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L and X ₁₄ =K,R.

In a more preferred embodiment of the variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO:8:

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L and X ₁₄ =K,R;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In an even more preferred embodiment of the second aspect, the compound according to the invention comprises or consists of an amino acid sequence according to SEQ ID NO:9 (cgtx 538).

In another preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -QCX ₇ -RX ₈ -CX ₉ -X ₁₀ -QTGCPYGKCMNRKCKCNRC (SEQ ID NO: 10);

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =A,Y,I,L,W,S,T,V,L,F; X ₈ =P,H,V,I,L,A; X ₉ =R,K,Q,N and X ₁₀ =K,D,A,R,E,V,L,I.

In another preferred embodiment of the variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO:10:

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =A,Y,I,L,W,S,T,V,L,F; X ₈ =P,H,V,I,L,A; X ₉ =R,K,Q,N and X ₁₀ =K,D,A,R,E,V,L,I;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a more preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -QCX ₇ -RX ₈ -CX ₉ -X ₁₀ -QTGCPYGKCMNRKCKCNRC (SEQ ID NO: 11);

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =A,Y,I,L,W; X ₈ =P,H,V,I,L,A; X ₉ =R,K,Q,N and X ₁₀ =K,D,A,R,E,V,L,I.

In a more preferred embodiment of the variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO:11:

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =A,Y,I,L,W; X ₈ =P,H,V,I,L,A; X ₉ =R,K,Q,N and X ₁₀ =K,D,A,R,E,V,L,I;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In an even more preferred embodiment of the second aspect, the compound according to the invention comprises or consists of an amino acid sequence according to SEQ ID NO: 12 (cgtx 539).

In another preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

X ₁ -X ₂ -CRX ₃ -X ₄ -X ₅ -QCYPHCX ₆ -X ₇ -X ₈ -TGCPYGKCMNRKCKCNRC (SEQ ID NO: 13);

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =T,G,S,N,I,K,Q,A,V,L,Y; X ₄ =P,S,T; X ₅ =R,K,P; X ₆ =R,K,Q,N; X ₇ =K,D,A,R,E,V,L,I and _X8 =E,Q,A,L,D,N,V,I.

In another preferred embodiment of the variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO: 13:

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =T,G,S,N,I,K,Q,A,V,L,Y; X ₄ =P,S,T; X ₅ =R,K,P; X ₆ =R,K,Q,N; X ₇ =K,D,A,R,E,V,L,I and _X8 =E,Q,A,L,D,N,V,I;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a more preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

X ₁ -X ₂ -CRX ₃ -X ₄ -X ₅ -QCYPHCX ₆ -X ₇ -X ₈ -TGCPYGKCMNRKCKCNRC (SEQ ID NO: 14);

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =T,G,S,N,I; X ₄ =P,S,T; X ₅ =R,K,P; X ₆ =R,K,Q; X ₇ =K,D,A,R and X ₈ =E,Q,A,L;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a more preferred embodiment of the variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO: 14:

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =T,G,S,N,I; X ₄ =P,S,T; X ₅ =R,K,P; X ₆ =R,K,Q; X ₇ =K,D,A,R and X ₈ =E,Q,A,L;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In an even more preferred embodiment of the second aspect, the compound according to the invention comprises an amino acid sequence according to SEQ ID NO: 15 (cgtx 540).

In another preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPYGKCMNRKCRCX ₁₄ -X ₁₅ -C (SEQ ID NO: : 16);

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,Q,T,S and X ₁₅ =G,R,K.

In another preferred embodiment of the variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO: 16:

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,Q,T,S and X ₁₅ =G,R,K;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a more preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPYGKCMNRKCRCX ₁₄ -X ₁₅ -C (SEQ ID NO: : 17);

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =Y,N and X ₁₅ =G,R.

In a more preferred embodiment of the variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO: 17:

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =Y,N and X ₁₅ =G,R;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In an even more particularly preferred embodiment of the second aspect, the compound according to the invention comprises or consists of an amino acid sequence according to SEQ ID NO: 18 (cgtx 541) or SEQ ID NO: 19 (cgtx 542).

In another preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

ISCX ₁ -X ₂ -X ₃ -X ₄ -X ₅ -CX ₆ -X ₇ -X ₈ -CX ₉ -X ₁₀ -X ₁₁ -TGCPYGKCMNRKCKCNRC (SEQ ID NO: 20);

where X ₁ =R,T,K,S,Y; X ₂ =T,G,S,N,I,K,Q,A,V,L,Y; X ₃ =P,S,T; X ₄ =R,K,P; X ₅ =D,Q,N,E; X ₆ =A,Y,I,L,W,S,T,V,L,F; X ₇ =D,R,P,K,E,S,T,Y; X ₈ =P,H,V,I,L,A; X ₉ =R,K,Q,N; X ₁₀ = _K ,D,A,R,E,V,L,I and X11 =E,Q,A,L,D,N,V,I.

In another preferred embodiment of the variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO: 20:

where X ₁ = R,T,K,S,Y; X ₂ =T,G,S,N,I,K,Q,A,V,L,Y; X ₃ =P,S,T; X ₄ =R,K,P; X ₅ =D,Q,N,E; X ₆ =A,Y,I,L,W,S,T,V,L,F; X ₇ =D,R,P,K,E,S,T,Y; X ₈ =P,H,V,I,L,A; X ₉ =R,K,Q,N; X ₁₀ = _K ,D,A,R,E,V,L,I and X11 =E,Q,A,L,D,N,V,I;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a more preferred embodiment of the second aspect, the compound according to the invention comprises or consists of the amino acid sequence:

ISCX ₁ -X ₂ -X ₃ -X ₄ -X ₅ -CX ₆ -X ₇ -X ₈ -CX ₉ -X ₁₀ -X ₁₁ -TGCPYGKCMNRKCKCNRC (SEQ ID NO: 21);

where X ₁ =R,T,K; X ₂ =T,G,S,N,I; X ₃ =P,S; X ₄ =R,K,P; X ₅ =D,Q,N,E; X ₆ =A,Y,I,L,W; X ₇ =D,R,P,K,E,S; X ₈ =P,H,V; X ₉ =R,K,Q; X ₁₀ =K,D,A,R and X ₁₁ =E,Q,A,L.

In a more preferred embodiment of a variant of the second aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO:21:

where X ₁ =R,T,K; X ₂ =T,G,S,N,I; X ₃ =P,S; X ₄ =R,K,P; X ₅ =D,Q,N,E; X ₆ =A,Y,I,L,W; X ₇ =D,R,P,K,E,S; X ₈ =P,H,V; X ₉ =R,K,Q; X ₁₀ =K,D,A,R and X ₁₁ =E,Q,A,L;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In an even more preferred embodiment of the second aspect, the compound according to the invention comprises or consists of an amino acid sequence according to SEQ ID NO: 22 (cgtx 545).

In a third aspect, the compound according to the invention comprises or consists of the amino acid sequence:

GVX ₁ -INVX ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPX ₁₄ -X ₁₅ -KCMNRKCX ₁₆ -CX ₁₇ -X ₁₈ -C (SEQ ID NO: 27);

where X ₁ =P,I,F,V,A,L,W; X ₂ =K,S,T,Y,R; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,S,T,Q; X ₁₅ =A,G,V,I,L; X ₁₆ =K,R, X ₁₇ =Y,N,Q,T,S and X ₁₈ =G,R,K.

In a variant of the third aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO:27:

where X ₁ =P,I,F,V,A,L,W; X ₂ =K,S,T,Y,R; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,S,T,Q; X ₁₅ =A,G,V,I,L; X ₁₆ =K,R, X ₁₇ =Y,N,Q,T,S and X ₁₈ =G,R,K;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a preferred embodiment of the third aspect, the compound according to the invention comprises or consists of the following amino acid sequence:

GVX ₁ -INVX ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPX ₁₄ -X ₁₅ -KCMNRKCX ₁₆ -CX ₁₇ -X ₁₈ -C (SEQ ID NO: 28);

where X ₁ =P,I,F; X ₂ =K,S; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =Y,N; X ₁₅ =A,G; X ₁₆ =K,R, X ₁₇ =Y,N and X ₁₈ =G,R.

In a preferred embodiment of the variant of the third aspect, the present invention also relates to a compound comprising or consisting of an amino acid sequence according to SEQ ID NO: 28:

where X ₁ =P,I,F; X ₂ =K,S; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =Y,N; X ₁₅ =A,G; X ₁₆ =K,R, X ₁₇ =Y,N and X ₁₈ =G,R;

wherein the compound is capable of selectively binding the potassium channel Kv1.3.

In a particularly preferred embodiment of the third aspect, the compound according to the invention has an amino acid sequence according to SEQ ID NO: 29 (cgtx 543), SEQ ID NO: 30 (cgtx 546) or SEQ ID NO: 31 (cgtx 548) contains or consists of

It is therefore particularly preferred that the compound according to the invention comprises or consists of an amino acid sequence according to SEQ ID NOs: 9, 12, 15, 18, 19, 22, 25, 26, 29, 30 or 31, Most preferred are compounds comprising or consisting of the amino acid sequence according to identification number: 25.

In some embodiments, 0 to 5, i.e. 0, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or insertions, provided that the compound is still able to selectively bind to and/or block or decrease the activity of the Kv1.3 potassium channel. should be able Compounds carrying such amino acid mutations are referred to as variants and also form part of the present invention.

Substitutions may be conservative or non-conservative substitutions, preferably substitutions are conservative substitutions. Conservative substitutions include substituting an amino acid for another amino acid that has similar chemical properties to the amino acid being substituted. In some embodiments, a substitution may also be an exchange of a naturally occurring amino acid for a non-natural amino acid.

The terms “amino acid deletion” and “amino acid insertion” are used herein according to their meanings common and well known in the art.

In another aspect, the present invention also provides a nucleic acid encoding an amino acid sequence according to the present invention.

In the context of the present invention, the term “nucleic acid” or “nucleic acid sequence” refers to a naturally occurring or synthetic deoxyribonucleotide or ribonucleotide polymer in single-stranded or double-stranded form capable of encoding a given amino acid sequence. The term may also differ from the original deoxyribonucleotide or ribonucleotide polymer in that one or more nucleotides of the original sequence are replaced by other nucleotides and/or are modified (chemically) by methods known to those skilled in the art. Derivatives of any deoxyribonucleotide or ribonucleotide polymer that can

It will be apparent to the skilled person that, due to the degeneracy of the genetic code, a given amino acid sequence according to the present invention may be encoded by different nucleotide sequences.

The invention also in a further aspect provides a vector comprising a nucleic acid sequence according to the invention.

Such vectors contain one or more nucleotide sequences according to the invention and are preferably any molecular vehicle designed for transfer between different host cells, such as, for example, a plasmid vector, a viral vector, a bacteriophage vector or any other vehicle. can In a preferred embodiment, said vector is a prokaryotic or eukaryotic expression vector.

As used herein, the term "expression vector" contains the desired coding sequence and the appropriate DNA sequences necessary for expression of the operably linked coding sequence, and contains a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. An expression vector may contain functional elements such as, for example, a promoter operably linked with the nucleic acid sequence to be transcribed, a termination sequence allowing proper termination of transcription, and a selectable marker. The person skilled in the art is aware that the nature of the promoter will depend on whether the vector will be used in a prokaryotic or eukaryotic host cell. In order to obtain stable expression for a long period of time, the expression vector may further comprise an origin of replication (ORI). Suitable expression vectors are known to the person skilled in the art. Depending on whether expression is to be achieved in a prokaryotic or eukaryotic host cell or in an in vitro expression system, the vector can be a prokaryotic and/or eukaryotic expression vector, such as a plasmid, cosmid, minichromosome, bacterial phage, retroviral vector, etc. have. The skilled person will be well aware of how to select an appropriate vector according to specific needs.

The invention, in another aspect, refers to a host cell comprising a nucleic acid sequence or vector according to the invention.

Depending on the field of application, the host cell may be a prokaryotic or eukaryotic host cell. Typical prokaryotic host cells are bacterial cells such as, for example, Escherichia coli [E. coli ( E. coli )]. Typical eukaryotic host cells are, for example, yeast cells such as for example Saccharomyces cerevisiae , insect cells such as for example Sf9/Sf21 cells, plant cells and mammalian cells such as for example For example, COS, CHO and HeLa cells.

The invention in a further aspect also relates to a pharmaceutical composition comprising a compound according to the invention.

In some embodiments, a pharmaceutical composition according to the present invention may comprise two or more of the compounds according to the present invention. For example, a pharmaceutical composition according to the invention may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 of the compounds according to the invention. In some preferred embodiments the pharmaceutical compositions of the present invention may comprise one or more compounds according to the present invention, wherein said compounds are SEQ ID NOs: 9, 12, 15, 18, 19, 22, 25, 26, 29, is selected from the group of compounds comprising or consisting of an amino acid sequence of 30 or 31.

Peptides considered particularly preferred throughout this disclosure for therapeutic use include those of SEQ ID NO: 25 (cgtx-544), SEQ ID NO: 29 (cgtx 543), and SEQ ID NO: 9 (cgtx 538) is a peptide. It should be noted that most of the experiments described below were performed using the peptide of SEQ ID NO: 25 (cgtx-544). However, the peptides of SEQ ID NO: 29 (cgtx 543), and SEQ ID NO: 9 (cgtx 538) were also tested for their selectivity compared to hERG and based on their sequence similarity, compared with that for cgtx 544 It seems reasonable to conclude that a similar effect can be observed for these peptides as well.

These peptides, as well as other peptides described herein, can be prepared by methods known in the art, such as solid phase synthesis. It should be noted that, as these peptides contain cysteine residues, active folding into their native state may be required to achieve optimal activity. Activity can be further enhanced by purifying the fully folded peptide. Folding can be achieved by oxidizing the synthesized peptide to achieve disulfide bridges between cysteine residues. This can be done by incubating the peptide in phosphate buffer in the presence of atmospheric oxygen, for example at a pH of about 8.0, as described in Example 8. Folding can be followed by mass spectrometry, since this folded peptide will exhibit a slightly reduced mass corresponding to the loss of hydrogen atoms during disulfide bridge formation. For cgtx 544 the difference in mass would be 4212 vs 4220.2 Da.

Purification of the fully folded peptide can be accomplished by methods known in the art, such as HPLC purification. A suitable approach is described in Example 8.

The pharmaceutical composition according to the invention may be in the form of, for example, inhalable powder pills, tablets, lacquered tablets, dragees, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions. It may be administered orally or may be administered rectally, for example, in the form of a suppository.

Administration may also be performed intranasally or sublingually.

Administration may additionally be effected parenterally, eg subcutaneously, intramuscularly or intravenously in the form of solutions for injection or infusion. Other suitable dosage forms are, for example, transdermal or topical administration, eg in the form of ointments, tinctures, sprays or transdermal treatment systems, or by inhalation in the form of nasal sprays or aerosol mixtures.

Dosage forms considered particularly preferred throughout this disclosure are intravenous, intramuscular or subcutaneous administration.

It is possible to use, for example, lactose, starch, such as corn starch or a starch derivative, talc, stearic acid or a salt thereof, and the like, for preparing pills, tablets, sugar tablets and hard gelatine capsules. Carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, semi-solid and liquid polyols, natural or hardened oils and the like. Suitable carriers for preparing solutions, for example injectable solutions, or emulsions or syrups, include, for example, water, physiological sodium chloride solution, alcohols such as ethanol, glycerol, polyols, sucrose, converted sugar, glucose, mannitol and vegetable oils. Pharmaceutical compositions may also contain additives such as fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavoring agents, perfuming agents, thickeners, diluents, buffer substances, solvents, solubilizers It may contain topical agents, agents to achieve a depot effect, salts to alter osmotic pressure, coating agents or antioxidants.

Examples of excipients suitable for a variety of different forms of the pharmaceutical compositions described herein can be found in "Handbook of Pharmaceutical Excipients", 2nd Edition, (1994), Edited by A Wade and PJ Weller.

In some embodiments the pharmaceutical composition may be a sustained release formulation.

In some embodiments the pharmaceutical composition according to the present invention may further comprise, in addition to at least one compound according to the present invention, other immunosuppressive agents suitable for the treatment of autoimmune diseases. Examples of such immunosuppressants include, for example, cortisol, hydrocortisol, dexamethasone, cyclophosphamide, nitrosourea, methotrexate, mercaptopurine, mitomycin C, bleomycin, mithramycin, cyclosporine, rapamycin, azathio prine, prednisone and deoxyspergualine and interferon.

In another preferred embodiment a separate composition comprising an additional immunosuppressant as described above may be administered in combination with a pharmaceutical composition comprising one or more compounds according to the invention to a mammal in need thereof.

In a further aspect the invention relates to a compound according to the invention or a pharmaceutical composition according to the invention for use in the treatment or prophylaxis of diseases associated with effector memory cells (T _EM -cells).

The present invention preferably relates to a compound according to the invention or a pharmaceutical composition according to the invention for use in the treatment of a disease selected from the group consisting of autoimmune diseases, obesity, periodontitis and/or tissue transplant rejection.

For the purposes of this disclosure, the term “treatment” refers to the curative amelioration of a particular disease, whereas the term “prevention” refers to prophylactic prophylaxis. It should be understood that both terms do not mean complete remission or prevention of the respective disease, but rather mean that there is an improvement compared to the situation in which the pharmaceutically active agent is not administered for therapeutic or prophylactic purposes.

In a particularly preferred embodiment the invention relates to the use of a compound according to the invention or a pharmaceutical composition according to the invention for use in the treatment or prophylaxis of autoimmune diseases.

The term “autoimmune disease(s)” in the context of the present invention refers to a disease state caused by an inappropriate immune response directed against a self-encoded entity, ie a self-antigen. Included within this definition are any number of disorders caused by deficiencies in the immune system that allow the body to attack its own tissues. In a preferred embodiment, the autoimmune disease is a T cell mediated autoimmune disorder.

Examples of autoimmune diseases that can be treated or prevented by the compounds and pharmaceutical compositions of the present invention include, for example, multiple sclerosis, rheumatoid arthritis, psoriasis, type 1 diabetes mellitus, vasculitis, Hashimoto's disease, asthma, atopic dermatitis, Autoimmune eye disease, Sjogren's syndrome, acute disseminated encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hepatitis, autoimmune oophoritis, celiac disease, Crohn's disease, gestational milk Pemphigus, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, Kawasaki disease, lupus erythematosus, myasthenia gravis, pre-myoclonic nystagmus syndrome, optic neuritis, Odd's thyroiditis, pemphigus, pernicious anemia, polyarthritis ( in dogs), primary biliary cirrhosis, Reiter's syndrome, Takayasu's arteritis, temporal arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis, ANCA-associated systemic vasculitis, Churg-Strauss syndrome, microscopic polyangiitis, colitis, inflammatory bowel disease , uveitis and psoriatic arthritis.

Some of these autoimmune diseases have been linked to T _EM -cells, such as ANCA-associated systemic vasculitis (Abdulahad, Nephrology , 2009 vol. 14 pp 26), Chug-Strauss syndrome, Wegener's granulomatosis, microscopic polyangiitis ( Berden, Arthritis & Rheumatism , 2009, vol.60, pp 1578), ankylosing spondylitis, Behcet's disease, colitis, Crohn's disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Sjogren's syndrome, type 1 diabetes mellitus (Ulivieri, Expert ) Rev. Vaccines , 2013 vol. 12 pp 297), systemic lupus erythematosus (Devarajan, Immunol . Res , 2013 vol. 57 pp 12), uveitis (Amadi-Obi, Nephrology , 2009, vol 14 pp 26), and psoriasis Arthritis (De Vlam, Acta ) Derm . Venereol , 2014, vol. 94, pp 627).

The same applies to obesity (Xu, Human Molecular Genetics , 2003, vol. 12, pp 551).

Given that cgtx 544 has been shown to act selectively on T _EM -cells and exhibit effects on rheumatoid arthritis, it is reasonable to assume that cgtx 544 and other peptides disclosed herein can be used to treat or prevent the disease. It seems reasonable.

In a preferred embodiment the autoimmune disease to be treated or prevented by the compounds and pharmaceutical compositions of the present invention is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, psoriasis, type 1 diabetes mellitus and vasculitis.

Transplantation of an organ or tissue into a new host is often due to a T cell mediated immune response against the new organ or tissue (eg, heart, liver, kidney, pancreas, or skin), resulting in a loss of the transplanted organ or tissue. may lead to rejection. Accordingly, the present invention also relates to a compound according to the invention or a pharmaceutical composition according to the invention for use in the treatment or prevention of organ or tissue transplant rejection. Organ transplant rejection has also been shown to be associated with T _EM -cells (see, eg, Macedo, Transplantation , 2012 vol 93 pp 813). Given the high selectivity of cgtx 544 and other peptides disclosed herein, it seems reasonable to assume that cgtx 544 and other peptides disclosed herein can be used to treat or prevent these diseases.

In another aspect the present invention relates to the treatment of an autoimmune disease, obesity, periodontitis and/or tissue transplant rejection in a mammal in need thereof, by administering to the mammal in need thereof a compound according to the present invention or a pharmaceutical composition according to the present invention. or how to prevent it.

Preferably the autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, psoriasis, type 1 diabetes and vasculitis.

The term “mammal” as used herein includes, for example, humans, non-human primates, mice, rats, rabbits, guinea pigs, dogs, cats, cattle, horses, sheep, pigs, goats, and the like. A preferred mammal is a human.

A unit dosage form of a pharmaceutical composition according to the present invention may contain any suitable effective amount of a compound according to the present invention in the same order as the daily dosage range for which it is intended to be employed.

A mammal in need of administration of a compound according to the invention or a pharmaceutical composition according to the invention, for example, is suffering from or at risk of developing a T cell mediated disease, preferably a T cell mediated autoimmune disease. It is a mammal with In a preferred embodiment said autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, psoriasis, type 1 diabetes and vasculitis.

The invention in a further aspect also relates to a method for preparing a compound according to the invention, a nucleic acid sequence according to the invention, a vector according to the invention or a pharmaceutical composition according to the invention.

The present invention also relates to:

(1) a compound comprising the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPX ₁₄ -X ₁₅ -KCMNRKCX ₁₆ -CX ₁₇ -X ₁₈ -C (SEQ ID NO: 1);

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,S,T,Q; X ₁₅ =A,G,V,I,L; X ₁₆ =K,R, X ₁₇ =Y,N,Q,T,S and X ₁₈ =G,R,K.

In a preferred embodiment, the compound is not HsTx 1 (SEQ ID NO: 32).

(2) a compound according to (1) comprising the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPX ₁₄ -X ₁₅ -KCMNRKCX ₁₆ -CX ₁₇ -X ₁₈ -C (SEQ ID NO: 2);

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =Y,N; X ₁₅ =A,G; X ₁₆ =K,R, X ₁₇ =Y,N and X ₁₈ =G,R.

In a preferred embodiment, said compound is not HsTx 1.

(3) a compound according to (1) comprising the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPNAKCMNRKCX ₁₄ -CX ₁₅ -X ₁₆ -C ( SEQ ID NO: 3);

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =K,R, X ₁₅ =Y,N,Q,T,S and X ₁₆ =G,R,K.

(4) a compound according to (3) comprising the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPNAKCMNRKCX ₁₄ -CX ₁₅ -X ₁₆ -C ( SEQ ID NO: 4);

where X ₁ =A,V,IX ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =K,R, X ₁₅ =Y,N and X ₁₆ =G,R.

(5) a compound according to (1) comprising the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPX ₁₄ -X ₁₅ -KCMNRKCX ₁₆ -CYGC ( SEQ ID NO: 5);

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,S,T,Q; X ₁₅ =A,G,V,I,L and _X16 =K,R.

(6) a compound according to (5) comprising the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPX ₁₄ -X ₁₅ -KCMNRKCX ₁₆ -CYGC ( SEQ ID NO: 6);

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =Y,N; X ₁₅ =A,G and X ₁₆ =K,R.

(7) a compound according to (1) comprising the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPNAKCMNRKCX ₁₄ -CYGC (SEQ ID NO: 7) ;

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I and X ₁₄ =K,R.

(8) a compound according to (7) comprising the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPNAKCMNRKCX ₁₄ -CYGC (SEQ ID NO: 8) ;

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L and X ₁₄ =K,R.

(9) The compound according to (8), comprising the amino acid sequence of SEQ ID NO: 9.

(10) a compound according to (1) comprising the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -QCX ₇ -RX ₈ -CX ₉ -X ₁₀ -QTGCPYGKCMNRKCKCNRC (SEQ ID NO: 10);

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =A,Y,I,L,W,S,T,V,L,F; X ₈ =P,H,V,I,L,A; X ₉ =R,K,Q,N and X ₁₀ =K,D,A,R,E,V,L,I.

(11) a compound according to (10) comprising the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -QCX ₇ -RX ₈ -CX ₉ -X ₁₀ -QTGCPYGKCMNRKCKCNRC (SEQ ID NO: 11);

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =A,Y,I,L,W; X ₈ =P,H,V,I,L,A; X ₉ =R,K,Q,N and X ₁₀ =K,D,A,R,E,V,L,I.

(12) The compound according to (11), comprising the amino acid sequence of SEQ ID NO: 12.

(13) a compound according to (1) comprising the amino acid sequence:

X ₁ -X ₂ -CRX ₃ -X ₄ -X ₅ -QCYPHCX ₆ -X ₇ -X ₈ -TGCPYGKCMNRKCKCNRC (SEQ ID NO: 13);

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =T,G,S,N,I,K,Q,A,V,L,Y; X ₄ =P,S,T; X ₅ =R,K,P; X ₆ =R,K,Q,N; X ₇ =K,D,A,R,E,V,L,I and _X8 =E,Q,A,L,D,N,V,I.

(14) a compound according to (13) comprising the amino acid sequence:

X ₁ -X ₂ -CRX ₃ -X ₄ -X ₅ -QCYPHCX ₆ -X ₇ -X ₈ -TGCPYGKCMNRKCKCNRC (SEQ ID NO: 14);

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =T,G,S,N,I; X ₄ =P,S,T; X ₅ =R,K,P; X ₆ =R,K,Q; X ₇ =K,D,A,R and X ₈ =E,Q,A,L.

(15) The compound according to (14), comprising the amino acid sequence of SEQ ID NO: 15.

(16) a compound according to (1) comprising the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPYGKCMNRKCRCX ₁₄ -X ₁₅ -C (SEQ ID NO: : 16);

where X ₁ =A,V,I,L; X ₂ =S,R,K,T,Y; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,Q,T,S and X ₁₅ =G,R,K.

(17) a compound according to (16) comprising the amino acid sequence:

X ₁ -X ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPYGKCMNRKCRCX ₁₄ -X ₁₅ -C (SEQ ID NO: : 17);

where X ₁ =A,V,I; X ₂ =S,R,K; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =Y,N and X ₁₅ =G,R.

(18) The compound according to (17), comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19.

(19) a compound according to (1) comprising the amino acid sequence:

ISCX ₁ -X ₂ -X ₃ -X ₄ -X ₅ -CX ₆ -X ₇ -X ₈ -CX ₉ -X ₁₀ -X ₁₁ -TGCPYGKCMNRKCKCNRC (SEQ ID NO: 20);

where X ₁ =R,T,K,S,Y; X ₂ =T,G,S,N,I,K,Q,A,V,L,Y; X ₃ =P,S,T; X ₄ =R,K,P; X ₅ =D,Q,N,E; X ₆ =A,Y,I,L,W,S,T,V,L,F; X ₇ =D,R,P,K,E,S,T,Y; X ₈ =P,H,V,I,L,A; X ₉ =R,K,Q,N; X ₁₀ = _K ,D,A,R,E,V,L,I and X11 =E,Q,A,L,D,N,V,I.

(20) a compound according to (19) comprising the amino acid sequence:

ISCX ₁ -X ₂ -X ₃ -X ₄ -X ₅ -CX ₆ -X ₇ -X ₈ -CX ₉ -X ₁₀ -X ₁₁ -TGCPYGKCMNRKCKCNRC (SEQ ID NO: 21);

where X ₁ =R,T,K; X ₂ =T,G,S,N,I; X ₃ =P,S; X ₄ =R,K,P; X ₅ =D,Q,N,E; X ₆ =A,Y,I,L,W; X ₇ =D,R,P,K,E,S; X ₈ =P,H,V; X ₉ =R,K,Q; X ₁₀ =K,D,A,R and X ₁₁ =E,Q,A,L.

(21) The compound according to (20), comprising the amino acid sequence of SEQ ID NO: 22.

(22) a compound comprising the amino acid sequence:

X ₁ -X ₂ -X ₃ -NVX ₄ -CX ₅ -X ₆ -X ₇ -X ₈ -X ₉ -CX ₁₀ -X ₁₁ -X ₁₂ -CX ₁₃ -X ₁₄ -X ₁₅ -TGCPX ₁₆ -X ₁₇ -KCMNRKCX ₁₈ -CX ₁₉ -X ₂₀ -C (SEQ ID NO: 23),

where X ₁ =T,Q,S,Y,N; X ₂ =I,F,V,A,L,W; X ₃ =I,T,Y,S,V,A,L; X ₄ =K,S,T,Y,R; X ₅ =R,T,K,S,Y; X ₆ =T,G,S,N,I,K,Q,A,V,L,Y; X ₇ =P,S,T; X ₈ =R,K,P; X ₉ =D,Q,N,E; X ₁₀ =A,Y,I,L,W,S,T,V,L,F; X ₁₁ =D,R,P,K,E,S,T,Y; X ₁₂ =P,H,V,I,L,A; X ₁₃ =R,K,Q,N; X ₁₄ =K,D,A,R,E,V,L,I; X ₁₅ =E,Q,A,L,D,N,V,I; X ₁₆ =Y,N,S,T,Q; X ₁₇ =A,G,V,I,L; X ₁₈ =K,R, X ₁₉ =Y,N,Q,T,S and X ₂₀ =G,R,K.

(23) a compound according to (22) comprising the amino acid sequence:

X ₁ -X ₂ -X ₃ -NVX ₄ -CX ₅ -X ₆ -X ₇ -X ₈ -X ₉ -CX ₁₀ -X ₁₁ -X ₁₂ -CX ₁₃ -X ₁₄ -X ₁₅ -TGCPX ₁₆ -X ₁₇ -KCMNRKCX ₁₈ -CX ₁₉ -X ₂₀ -C (SEQ ID NO: 24);

where X ₁ =T,Q; X ₂ =I,F; X ₃ =I,T; X ₄ =K,S; X ₅ =R,T,K; X ₆ =T,G,S,N,I; X ₇ =P,S; X ₈ =R,K,P; X ₉ =D,Q,N,E; X ₁₀ =A,Y,I,L,W; X ₁₁ =D,R,P,K,E,S; X ₁₂ =P,H,V; X ₁₃ =R,K,Q; X ₁₄ =K,D,A,R; X ₁₅ =E,Q,A,L; X ₁₆ =Y,N; X ₁₇ =A,G; X ₁₈ =K,R, X ₁₉ =Y,N and X ₂₀ =G,R.

(24) The compound according to (23), comprising the amino acid sequence of SEQ ID NO:25 or SEQ ID NO:26.

(25) a compound comprising the amino acid sequence:

GVX ₁ -INVX ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPX ₁₄ -X ₁₅ -KCMNRKCX ₁₆ -CX ₁₇ -X ₁₈ -C (SEQ ID NO: 27);

where X ₁ =P,I,F,V,A,L,W; X ₂ =K,S,T,Y,R; X ₃ =R,T,K,S,Y; X ₄ =T,G,S,N,I,K,Q,A,V,L,Y; X ₅ =P,S,T; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W,S,T,V,L,F; X ₉ =D,R,P,K,E,S,T,Y; X ₁₀ =P,H,V,I,L,A; X ₁₁ =R,K,Q,N; X ₁₂ =K,D,A,R,E,V,L,I; X ₁₃ =E,Q,A,L,D,N,V,I; X ₁₄ =Y,N,S,T,Q; X ₁₅ =A,G,V,I,L; X ₁₆ =K,R, X ₁₇ =Y,N,Q,T,S and X ₁₈ =G,R,K.

(26) a compound according to (25) comprising the amino acid sequence:

GVX ₁ -INVX ₂ -CX ₃ -X ₄ -X ₅ -X ₆ -X ₇ -CX ₈ -X ₉ -X ₁₀ -CX ₁₁ -X ₁₂ -X ₁₃ -TGCPX ₁₄ -X ₁₅ -KCMNRKCX ₁₆ -CX ₁₇ -X ₁₈ -C (SEQ ID NO: 28);

where X ₁ =P,I,F; X ₂ =K,S; X ₃ =R,T,K; X ₄ =T,G,S,N,I; X ₅ =P,S; X ₆ =R,K,P; X ₇ =D,Q,N,E; X ₈ =A,Y,I,L,W; X ₉ =D,R,P,K,E,S; X ₁₀ =P,H,V; X ₁₁ =R,K,Q; X ₁₂ =K,D,A,R; X ₁₃ =E,Q,A,L; X ₁₄ =Y,N; X ₁₅ =A,G; X ₁₆ =K,R, X ₁₇ =Y,N and X ₁₈ =G,R.

(27) The compound according to (26), comprising the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31.

(28) capable of selectively binding potassium channel Kv1.3, (1), (2), (3), (4), (5), (6), (7), (8), (9) ), (10), (11), (13), (14), (17), (19), (20), (22), (23), (25) or (26) according to compound.

(29) capable of selectively binding potassium channel Kv1.3 when compared to potassium channel Kv1.1, (1), (2), (3), (4), (5), (6), (7) ), (8), (9), (10), (11), (13), (14), (17), (19), (20), (22), (23), (25) or The compound according to any one of (26).

(30) A nucleic acid sequence encoding an amino acid sequence as recited in any one of (1) to (27).

(31) A vector comprising the nucleic acid sequence according to (30).

(32) A host cell comprising the nucleic acid sequence according to (30) or the vector according to (31).

(33) A pharmaceutical composition comprising a compound according to any one of (1) to (29).

(34) A compound according to any one of (1) to (29) or a pharmaceutical composition according to (33) for use in the treatment of an autoimmune disease, obesity, periodontitis and/or tissue transplant rejection.

(35) A compound according to any one of (1) to (29) or a pharmaceutical according to (33) for the manufacture of a medicament for treating or preventing autoimmune disease, obesity, periodontitis and/or tissue transplant rejection. Use of the composition.

(36) administering the compound according to any one of (1) to (29) or the pharmaceutical composition according to (33) to a mammal in need thereof, thereby causing autoimmune disease, obesity, periodontitis and/or A method of treating or preventing tissue transplant rejection.

(37) A method for producing a compound according to any one of (1) to (29), using the nucleic acid sequence according to (30), the vector according to (31) or the host cell according to (32).

Example

Example 1 - Electrophysiological measurements

Potassium currents were measured by using Spodoptera frugiperda (Sf21) cells expressing human Kv1.3 (hKv1.3) channels. This channel activity is induced upon membrane depolarization to voltages more positive than -40 mV. Activation kinetics is rapidly and strongly voltage dependent, whereas inactivation is much slower and exhibits no significant voltage dependence. In Sf21 cells, hKv1.3 has an average current amplitude of 250 pA - 5 nA at +40 mV.

On days 1-3 post-infection, using a Port-A-Patch (Nanion Technologies GmbH), potassium currents were recorded with planar patch clamp technique. The ion current was activated by a voltage step to a depolarization potential of +40 mV for 200 ms.

Sf21 cells were treated with Grace supplemented with 10% FBS (fetal bovine serum), 2 mM glutamine, 1x Yeastolate (derived from 50x stock solution) and 0.1% Pluronic® F68 (BASF). ) cultured in insect cell medium to a cell density of 2 - 3 x 10 ⁶ cells/ml.

Electrophysiological buffers are as follows:

External Buffer: 160 mM NaCl

4.5 mM KCl

1 mM MgCl ₂

2 mM CaCl ₂

5 mM D-glucose monohydrate

10 mM HEPES/NaoH pH 7.4.

Internal Buffer: 75 mM KCl

10 mM NaCl

70 mM K-fluoride

2 mM MgCl ₂

10 mM EGTA

10 mM HEPES/KOH pH 7.2.

Binding of cgtx-544 to Kv1.3, Kv1.1 and Kv1.5 to demonstrate the selectivity of the peptides described herein (SEQ ID NOs: 1-31) and in particular cgtx-544 (SEQ ID NO: 25) The IC50 for HsTx 1 (SEQ ID NO: 32) was tested as a control.

Peptides were dissolved at 1 μg/μl in 20 mM NaPO ₄ buffer pH 8.2 (1 mg in 1 ml folding buffer), 3x 1 L of same buffer as above, Float-a-Lyzer cut off 500 Da at room temperature Dialysis against [Spectrapor].

HsTx 1 binding was tested at various concentrations, eg, 100 pM, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, and 1 μM, depending on the channel tested. Representative measurements for Kv 1.1, Kv1.3 and Kv1.5 are shown in FIGS. 1 a), 1 b), and 1 c), respectively.

The IC50 for HsTx 1 binding to Kv1.3 was 25 ± 2.1 nM. The IC50 for HsTx 1 binding to Kv1.1 was 11.3 μM. No binding could be detected for Kv1.5. Thus, HsTx 1 has about 450-fold higher selectivity for Kv1.3 compared to Kv1.1.

Cgtx-544 binding was tested at various concentrations, eg, 1 nM, 10 nM, 50 nM, 100 nM, 1 μM, and 5 μM, depending on the channel tested. Representative measurements for Kv1.1, Kv1.3 and Kv1.5 are shown in FIGS. 1 d), 1 e), and 1 f), respectively.

The IC50 for cgtx-544 binding to Kv1.3 was 6.9 nM. No binding to Kv1.5 could be detected up to a concentration of 10 μM. Kv1.1 was blocked by about 22% at 10 μM. Therefore, cgtx-544 has a higher selectivity for Kv1.3 compared to Kv1.1 when compared to HsTx 1.

Example 2 - Kv1 .3 and hERG comparison of binding to

In the examples below, binding of selected peptides to Kv1.3 and hERG was compared by electrophysiological measurements.

cell system

To determine binding to Kv1.3, Jurkat cells endogenously expressing Kv1.3 were used. For binding to hERG, HEK cells stably expressing hERG were used.

cell culture

Cell culture was performed using standard cell culture techniques. Briefly, cells were grown on 10 cm culture dishes or T75 culture flasks in DMEM-based medium. Cells were split every 2-3 days, using a density of about 60-80%. For recording on a patchliner (Nanion), Jurkat cells were resuspended and then spun in a centrifuge. Prior to spinning, HEK cells were trypsinized to detach the cells from the dish surface. After spinning, cells were resuspended in external recording buffer to a density of approximately 1-2 million cells per ml.

Internal recording solution: 50 mM KCl

10 mM NaCl

60 mM KF

20 mM EGTA

10 mM HEPES/KOH, pH 7.2, (Osm: about 290 mOsm).

5 mM Mg-ATP and 0.3 mM Na-GTP were freshly added to the inner solution, and the pH was adjusted to 7.2.

External recording buffer: 140 mM NaCl

4 mM KCl

1 mM MgCl2

2 mM CaCl2

5 mM D-glucose monohydrate

10 mM HEPES/NaOH pH 7.4, (Osm: about 298 mOsm).

compound

The following compounds were tested:

Compound 1: cgtx 538

Compound 2: cgtx 539

Compound 3: cgtx 540

Compound 4: cgtx 541

Compound 5: cgtx 542

Compound 6: cgtx 543

Compound 7: cgtx-544

Compound 8: cgtx 547.

Electrophysiological measurements

Electrophysiological recordings were performed using a patchliner that simultaneously recorded up to 8 channels. The patchliner is an automated patch clamp system that utilizes a borosilicate glass chip (NPC-16) for flat patch clamps and operates through the PatchControlHT software. An NPC-16 chip with a resistance of 3-5 MΩ for Jurkat cells and 2-3 MΩ for HEK cells was used for recording. During recording, cells were placed in a "cell hotel" where they were periodically pipetted up and down to maintain viability. Cells survived for approximately 2-3 hours.

patch control HT software

To run the patchliner, run two programs simultaneously on the computer - Nanion's PatchControlHT and PatchMaster (HEKA).

The patchmaster controls the HEKA EPC10 amplifier and performs the recording. Pulse protocols and online analysis can be created within these programs. Using PatchControlHT, a complete experimental routine is defined from capturing and sealing cells to obtain the desired recording to go to whole cells. With two-way communication between PatchControlHT and the Patchmaster, PatchControlHT can adjust its behavior according to the cell state (giga seal, whole cell composition).

The program fully explores the robotic capabilities of the patchliner, including pipettes performing solution exchanges. At the same time, the patch control HT also controls the amplifier and reads all important patch parameters from the patch master. PatchControlHT is a graphical user interface where all parameters and success criteria for the experiment are defined. If the success criterion does not reach a recording well, the option for PatchControlHT to stop recording from that particular well is possible.

experiment routine

To establish proper recording conditions, chip filling, cell capture, sealing, and entry into whole cell mode were performed by standard procedure functions of the patchliner. At the end of this routine, the amplifier's environment (eg holding, holding potential, slow capacitive compensation and gain) was properly set up for the desired recording and then recording began.

Within each experiment, cells were washed 3 times with external solution to ensure stable recording. Compounds 1-8 were further tested on hERG expressing cells. The exposure time of each compound was 4 minutes in total, and each experiment was performed for 24 minutes.

voltage protocol

For hERG: hERG current using voltage step protocol from holding potential of -80 mV to -40 mV for 500 ms, then step to +40 mV for 500 ms, -40 mV for 500 ms to obtain tail current After using the test pulse, it was induced by returning to the holding potential. This step was repeated every 10 seconds.

The protocol was continuously performed and online results were continuously read. By doing this, changes due to washing and/or compound application could be monitored and the attainment of steady state could be assessed.

data analysis

Data recording and analysis were performed with 2x EPC10 Quadro amplifiers, patchmaster software package [HEKA Electronics; Lambrecht/Paltz, Germany], Excel and Igor [WaveMetrics; USA). ] was used.

For pharmacology for hERG, the peak amplitude at the second -40 mV step (tail current) was calculated using the online analysis function in Patchmaster. The actual peak was calculated by subtracting the calculated leakage from the first step to -40 mV from the peak, which could be plotted as a function of time.

The values were exported to Excel to calculate Igor's concentration response curve. Igor (Wavemetrics; USA) was used to extract and export the figures. Concentration response curves were constructed in Igor. Hill's equations were fitted to estimate the IC50 for each compound, and the IC50 was calculated relative to the maximum block. Values are expressed as mean ± S.E.M where possible.

result

hERG about current tested compounds 1-8

Electrophysiological measurements of compounds 1-8 against Kv1.3 currents indicated that these peptides were active. Therefore, compounds 1-8 were tested on hERG expressing HEK cells. To investigate the effects of these compounds, a two-point screening procedure was performed. Typically, 3 to 5 cells were tested to ensure accuracy. Each experiment was started with 3 washes to ensure a stable current. The hERG current increased slightly, but became stable after the second wash. Compounds 1, 2, 4, 5, 7, and 8 appeared to have only a low effect on hERG current. Typically, the effects of compounds 1-8 could also be explained by the run-down effect of hERG currents. The results are presented in FIG. 3 . A summary of the mean current block ± SEM of all compounds is presented in Table 1.

<Table 1>

Table 1: Summary of effects of compounds 1-8. The test concentration is different for each compound. Normalized blocking was calculated from the steady-state current at the end of each compound application. All compounds appeared to have no effect or only a small effect on hERG current at lower concentrations. At higher concentrations, the compounds appeared to affect hERG, but these effects could be due to run-down of hERG currents.

further analysis

The analysis was performed after applying the folding protocol as described in Example 8 to each peptide, even without purification of the fully folded peptide.

In addition, cgtx-544 was folded and purified as mentioned above. The assay was then repeated using this cgtx-544 (Sing). In addition, cgtx-544 (Sing) was then tested on Kv1.3 by incubating for a long period of time, i.e., about 20 minutes (a time that better correlated with the longer rate of action of the peptide). Under these conditions cgtx-544(Sing) displays an IC50 of about 900 pM after folding and purification compared to 6.9 nM of unpurified cgtx-544 (see Example 1). The results are shown in FIG. 35 .

Example 3 - human and rat in plasma cgtx -544 (SEQ ID NO: 25) of stability

1. Human and rat in serum samples ex vivo Determination of stability

The goal was to test the stability of the cgtx-544 peptide in mammalian plasma after different incubation times at room temperature (RT) and 37°C. Stability was defined as the IC50 value of cgtx-544 after different incubation times at RT and 37°C. Plasma was prepared from fresh blood samples from healthy rats or humans. Heparin was added to fresh blood samples to prevent clogging. Plasma preparation was performed by centrifugation at 2000×g for 2 min at RT.

The IC50 of cgtx-544 was defined before the start of the experiment—electrophysiological assays yielded an IC50 value of 46.1 nM ± 3.9 nM (for n=5 cells). Because the cgtx-544 peptide used in these experiments was incompletely folded, a higher than normal IC50 was determined. The prepared negative control of human and rat plasma did not show any non-specific binding of plasma components to Kv1.3 channels expressed in Sf21 insect cells.

To investigate stability and activity in plasma, the peptides were added to freshly prepared human and rat plasma at a final concentration of 20 μM. The sample was split into two aliquots and incubated at 37° C. and room temperature (RT, 20° C.). For electrophysiological analysis, serial dilutions of the samples were prepared.

Time point 0 h : Immediately after the addition of the peptide to the plasma, samples were analyzed which indicated a strong affinity for the Kv1.3 channel. The IC50 value for cgtx-544 in human plasma improved to 20.9 nM and the IC50 value in rat plasma improved to 15.0 nM (data not shown).

Time point 24 h : For the next 24 h, no significant change in activity of the stored peptide probes at 37° C. and RT could be detected.

Time point 48 h : After 48 h, the activity of the peptides incubated at 37° C. decreased, and the IC50 value of cgtx-544 incubated in human plasma or rat plasma was 2-fold higher compared with t=0 h (37.3 nM each and 57.8 nM). The activity of cgtx-544 stored at RT remained unchanged.

Time point 14 days : No activity was detected after 14 days of incubation at 37°C. The cgtx-544 probe incubated at RT displayed unchanged activity.

Example 4 - isolated human T _EM on the cell cgtx -544 (SEQ ID NO: 25) Efficacy

One. isolated human T _EM at Kv1 .3 analysis of currents;

The insect cell line Spodoptera frugiperda (Sf21) was used in combination with a baculovirus infection system for heterologous expression of recombinant ion channels. Sf21 cells confer highly specific protein expression and precise post-translational modifications, creating functional channels with the proposed comparability of electrophysiological parameters of ion channels in vivo. Therefore, it was necessary to compare the electrophysiological properties of the potassium-selective ion channel Kv1.3 expressed in Sf21 cells with the electrophysiological properties of the Kv1.3 channel endogenously expressed in T _EM cells.

To compare the characteristic voltage-dependent Kv1.3 channel activation, a typical voltage protocol from -60 mV to +60 mV in 20 mV steps (activation pulse 200 ms) was used. Cells were clamped at a holding potential of -100 mV. Figure 4 presents the current response (Figures 4a and 4b, respectively) and pulse protocol (Figure 4c) for voltage-dependent Kv1.3 activation of two individual Sf21 and T _EM cells.

Figure 5 presents the corresponding normalized voltage-current-correlation. Kv1.3 begins to open at the depolarization of the membrane from the holding potential (-100 mV) to -20 mV, and reaches its maximum open state at +60 mV. Even though the expression of Kv1.3 is much higher in Sf21 cells, its characteristic voltage-dependent activation remains unchanged.

Obviously, the maximum observed current resulting from Kv1.3 activity, and the applied voltage, allow us to evaluate the overall conductivity of all Kv1.3 channels. Therefore, the number of channels can be extracted using the known conductivity of a single Kv1.3 channel. The maximum K+ outward current is defined as the difference between the active but unblocked current and the maximally blocked (completely blocked) current during the first 20% of the current trace. By fluctuating the infection conditions, it is possible to control the expression level of Kv1.3 in Sf21 cells according to the need for investigation. Under our standard conditions, a maximum current of 2 - 5 nA, a conductivity of 33 - 50 nS and a number of 2500 - 3800 channels per cell can be evaluated.

The heterologous expression system in Sf21 cells represents a suitable method for the electrophysiological analysis of pharmacologically interesting ion channels in vitro. Based on these observations, it can be inferred that folding of Kv1.3 ion channels and inserting them into the plasma membrane of Sf21 cells is almost equivalent to native Kv1.3 protein in T _EM cells.

It could be clearly demonstrated that heterologous expressed Kv1.3 channels could serve as a model system to study drug targeting by evaluating electrophysiological parameters. Specific Kv1.3 blockade could be accomplished using the peptide compound cgtx-544. These peptides show high affinity for the Kv1.3 channel, as presented above and as tested on isolated human T _EM cells ( FIG. 6 ). It should be noted that the cgtx-544 used in these experiments was incompletely folded and exhibited a higher than normal IC50. Nevertheless, the compound showed the same values on Kv1.3 transfected Sf21 cells as on human T _EM cells ( FIG. 6 ).

Example 5 - peptide cgtx -544 (SEQ ID NO: 25) Therapy for antigen-induced arthritis using

1. Experimental model

1. AIA - antigen-induced arthritis

A disease model in rats Antigen-induced arthritis (AIA) exhibits a T cell dependent autoimmune arthritis that resembles many features of rheumatoid arthritis in humans. Thus, such a system can serve as a disease model for evaluating the therapeutic success of the T cell inhibitory peptides disclosed herein. One major advantage of this therapeutic approach over current therapies (e.g., using systemic cytostatic agents) is that only a small subset of the immune system that is causal to the development of the disease is suppressed, whereas For other important components of the immune defense, such as the innate immune system, is the fact that it is still intact. In addition, these disease models allow testing of the in vivo efficacy and immune tolerance of cgtx-peptides.

2. Induction of Arthritis

The entire experiment takes place within a period of 4 weeks. Pre-immunization was performed with mBSA and M. in incomplete Freund's adjuvant (IFA). Subcutaneous injection of a mixture of tuberculosis preparations takes place on days -21 (test start date) and day-14. Local inflammation induction in one knee joint is induced by a single intra-articular injection of the antigen mBSA into the right knee on day 0. In this experimental setup, the second (left) untreated knee joint serves as a control within the subject.

A severe swelling reaction develops in the treated knee joint during the first few hours after induction, typically reaching maximum swelling on days 1 and 2 post induction. This phenotype is stable for an additional 2-3 days, after which the swelling is relieved again.

This scheme allows the induction of localized recurrent arthritis in >95% of laboratory animals. In the negative control experiment, the same volume (as for the mBSA induction solution) of 0.9% NaCl was administered to the knee of the control animal (see Figures 7 and 8).

2. Substances and methods

2.1 Animals

Female Lewis rats of approximately 8 weeks of age with a body weight (BW) of 180-200 g [Janvier (France)]. The animals were placed under normal breeding conditions - 5 animals per Makrolon cage type IV; Unlimited supply of food and water in a 12-hour day/night rhythm; room temperature at 22 +/- 2°C; Air humidity under 55 +/- 5%.

2.2. Generation of Emulsions for Immunization, Animal Immunization & Induction of Local Arthritis

2.2.1. Emulsions and Immunization Procedures for Immunization

2.2.1.1. Substance: Mycobacterium tuberculosis H37 Ra (BD 231141)

Incomplete Freund's Adjuvant [Sigma F5506]

mBSA (Sigma A1009-1G)

0.9% NaCl [Brown]

Isoflurane [Abbott].

2.2.1.2. Pre-immunization emulsions:

Solution A: mBSA stock solution (50 mg/ml):

20 ml aqueous injection is added to one vial mBSA (1 g) under aseptic conditions. The sterile solution can be stored at 4° C. for several months.

Solution B: Complete Freund's Adjuvant (CFA) Stock Solution (10 mg/ml):

10 ml (1 vial) of incomplete Freund's adjuvant, M. containing 100 mg bacteria. Add to 1 vial of tuberculosis. This solution can be stored at 4° C. for 1 month.

Preparation of the mixture:

When both components are mixed in equal volumes, an emulsion is created (mixed under aseptic conditions on a clean bench). The components are pipetted into 15 ml or 50 ml Falcon tubes as described below:

1. Solution A - mBSA Stock Solution

2. Solution B - M. tuberculosis solution.

Immediately after adding the ingredients, the tube is vortexed for 20 seconds. In the final preparation step, the solution is emulsified using a sonicator (0.5 cycles with 60% amplitude) until a thick emulsion is formed.

2.2.1.3. pre-immunization

The animals were pre-immunized twice prior to the final arthritis induction. Pre-immunizations were made on days-21 and 14. Immunization solutions were injected subcutaneously at these time points. After induction of anesthesia using 5% isoflurane, anesthesia was maintained with 2% isoflurane. A total of 500 μl of the emulsified solution was applied by using a 25 G cannula. 125 μl of the pre-immunization emulsion was injected subcutaneously into the two sides of the tail mechanism (right and left) as well as the two sides above the left and right scapula.

2.2.1.4. Induction of local arthritis

On day 0, arthritis was induced by intra-articular administration of antigen (500 μg mBSA in 50 μl 0.9% NaCl) to the right knee joint. The left knee was left untouched and was used as a control within the subject. The animals were anesthetized and the injection site disinfected. This application was performed with a short, fine cannula (30 G, 1/2) to minimize tissue damage. After the antigen was administered and the cannula was withdrawn, the joint was extended with several slow flexions.

2.2.2. Measurement of swelling of the knee joint

The sagittal diameter of the knee joint was determined with a caliper. Laboratory animals were anesthetized for a short period and placed posteriorly. The legs were adjusted to a 90 degree angle, and measurements of the sagittal diameter of the knee were performed three times.

2.2.3. blood collection

For blood collection, the animal is briefly anesthetized, the tail is disinfected, the tail vein is cannulated, and 50 μl of blood is collected using a heparin-coated pipette, which is then mixed with 5 μl of a stabilizer ( CPD) and 1 μl heparin (1.5 U/μl diluted 1:4 in 0.9% NaCl).

2.2.3. hematology

White blood cells (WBC) were counted in an automated manner using a Coulter counter.

2.2.4. measurement of body weight

The body weight of the experimental animals was measured once a day between 8:00 and 9:00 AM.

2.2.5. Clinical examination and measurement of swelling of the knee joint

After induction of focal arthritis, laboratory animals were examined daily. Swelling of the knee joint was measured and general health was scored.

2.3. Treatment of laboratory animals

Treatment of the animals was started on day -3 with administration of peptide cgtx-544 (SEQ ID NO: 25) at 1 mg/kg body weight (BW) in 0.6 ml volume of 0.9% NaCl, or alternatively control For animals, treatment was started with the administration of an equal volume of 0.9% NaCl (excipient). Each of the peptides or excipients was administered i.v. administered. Animals thus treated were observed for approximately 1 hour after treatment.

3. Results

During the pre-immunization phase, laboratory animals were intensively monitored and blood samples were taken on days-21,-14, -7, 0 and 7 to monitor the animal's immune status. . There was a very good correlation between the incidence of inflammation and the counts of white blood cells (WBCs, leukocytes) in the peripheral blood at the corresponding time points of analysis.

The number of WBCs in the peripheral blood of recently immunized animals (days-21 and 14) is significantly increased when compared to untreated animals (see FIG. 9 ). The number of WBCs reaches a maximum between Days 14 and 7 and drops thereafter. An increase in WBC correlates with the severity of inflammation induced by immunization.

Immediately after induction of focal arthritis, WBC shows a short-term increase (see FIG. 9 ). However, this short-term increase is smaller because the inflammatory response is localized. Control animals immunized with 0.9% NaCl did not show any difference in WBC counts.

In pre-immunized animals, a severe swelling reaction of the right knee joint develops a few hours after induction of local arthritis by mBSA-injection (see FIG. 8 ). In contrast, the untreated left knee, serving as the control knee in the subject, does not show any signs of a swelling response.

Comparing animals treated with cgtx-544 peptide with untreated (excipient) controls, it was found that cgtx-544 treatment resulted in a clear reduction in knee joint swelling compared to untreated control animals (see Figure 10). ). It can be concluded that animals treated with cgtx-544 show a clearer reduction in knee joint swelling and more rapid regression of swelling compared to untreated control animals.

Example 5a - RA AIA rat in the model cgtx Prophylactic Efficacy of -544 Peptide

1. Materials and Methods

After two immunizations on days-21 and 14 with an emulsion of 750 μg heat inactivated Mycobacterium (M.) tuberculosis and antigen mBSA in complete Freund's adjuvant, in Example 5 AIA was induced by intra-articular injection of methylated BSA into the right knee on day 0 as described. For cgtx-544 peptide efficacy studies, rats were given 0.1, 1 or 5 mg/kg cgtx-544 peptide (low, medium and high dose levels) by intravenous injection or vehicle (0.9% NaCl) starting on day -3. was administered daily for 10 days (days -3 to 6).

Arthritis severity was monitored by measuring the knee joint diameter in triplicate using a caliper. Values were normalized to Day-21. The difference in knee swelling values was calculated by subtracting the left (non-induced) knee diameter from the right (induced) knee diameter.

To monitor hematological parameters, blood samples were taken regularly. Hematological parameters were assessed in all animals weekly before induction, on the day of induction and 10 days after induction (days-21, 14, -7, 0 and 10). Hematological data of untreated animals (day-21) were almost equivalent to those reported in the literature (Waynforth & Flecknell, 1992). In particular, absolute counts of leukocytes (WBC), neutrophil granulocytes and lymphocytes were analyzed, the last two were also referred to as percentages of total WBC. EDTA leukocytes from 7 subjects from both vehicle and cgtx-544 peptide therapy groups were measured with a Sysmex XT-2000i.

2. Results

cgtx-544 (0.1, 1, and 5 mg/kg BW) at all dose levels tested showed a clearly reduced, dose-dependent and statistically significant effect on knee swelling in treated animals ( FIG. 18 ). .

For statistical analysis of the results, Welsh's two-sample t-test was used. Next, statistical analyzes of cgtx-544 (0.1, 1, and 5 mg/kg BW) of all dose levels tested are presented on days 1 and 3. All dose levels tested showed a statistically significant reduction in knee swelling when compared to vehicle control ( FIG. 19 ).

In summary, cgtx-544 peptide therapy in the AIA model showed a potent effect on inflammatory arthritis symptoms, particularly knee swelling, with a reduction of up to 70%. After 7 days of treatment, the knee diameter in the therapy group was further reduced to a diameter approximately equivalent to that of the control knee in the non-induced subjects. Moreover, WBCs, neutrophil granulocytes and lymphocytes (data not shown) of cgtx-544 peptide-treated subjects exhibited absolute and relative cell counts on par with vehicle-treated subjects.

Example 5B - RA's AIA rat in the model cgtx -544's healing efficacy

1. Materials and Methods

After two immunizations on days-21 and 14 with the antigen mBSA and 750 μg heat inactivated emulsion of Mycobacterium (M.) tuberculosis in complete Freund's adjuvant, Example 5 above AIA was induced by intra-articular injection of methylated BSA into the right knee on day 0 as described in For cgtx-544 peptide efficacy studies, rats received daily intravenous injections of 1 mg/kg cgtx-544 peptide starting on day 0 or starting on day 1 immediately after arthritis induction. Control animals received daily injections of vehicle (0.9% NaCl).

Arthritis severity was monitored by measuring the knee joint diameter in triplicate using a caliper. Values were normalized to Day-21. The difference in knee swelling values was calculated by subtracting the left (non-induced) knee diameter from the right (induced) knee diameter.

2. Results

Initiation of treatment at both test time points with cgtx-544(Mix) (1 mg/kg BW) showed a clearly decreasing and statistically significant effect on knee swelling in the treated animals ( FIG. 20 ).

For statistical analysis of the results, Welsh's two-sample t-test was used. Next, statistical analyzes of both treatment regimens (d0 and d1) of cgtx-544 are presented for Days 1, 2 and 3. Both treatment regimens showed a statistically significant reduction in knee swelling when compared to vehicle control ( FIG. 21 ).

White blood cell (WBC) counts over time are presented in FIG. 22 . Neutrophils over time are presented in FIG. 23 . Lymphocytes over time are shown in FIG. 24 .

cgtx-544 (Mix) treatment showed a significant reduction in knee joint swelling. Residual swelling in treated animals was presumed to be due to antibody/antigen responses. Therefore, immunized as well as naive animals were tested for the presence of mBSA specific antibodies. Antibodies against mBSA could be detected in all immunized animals of groups H and I 7 days after arthritis induction ( FIG. 27 ). Naive animals (Experimental group F) did not show any reactivity to mBSA. cgtx-544 therapy does not appear to inhibit B-lymphocyte and antibody formation. Additionally, animals immunized after 10 days on cgtx-544 peptide therapy showed no reactivity to cgtx-544(Mix) (fusion protein of L544-2a with trx-tag, data not shown).

In summary, cgtx-544 peptide therapy in the AIA model showed a strong effect on inflammatory arthritis symptoms, particularly knee swelling, with up to 40% reduction or reduction of up to 40% when therapy was started on day 0 immediately after arthritis induction. Reductions of up to 58% were seen when therapy was started on day 1. After 7 days of treatment, the knee diameter in the therapy group was further reduced to a diameter approximately equivalent to that of the control knee in the non-induced subjects. Moreover, the WBCs, neutrophil granulocytes and lymphocytes of the cgtx-544 peptide-treated subjects exhibited absolute and relative cell counts almost equivalent to those of the vehicle-treated subjects.

Example 5c - RA AIA rat in the model cgtx -544 (Mix) sustained efficacy

Previous experiments (see Examples 5A and 5B) demonstrated that cgtx-544 had superior efficacy in the AIA rat model when different treatment regimens - curative and prophylactic - were applied daily. We sought to clarify how long the activity of cgtx-544 lasts in the AIA model in order to obtain an indication of how long the treatment interval with cgtx-544 will last. Therefore, a therapeutic treatment regimen was established according to the once-weekly application plan.

1. Materials and Methods

After two immunizations on days-21 and 14 with the antigen mBSA and 750 μg heat inactivated emulsion of Mycobacterium (M.) tuberculosis in complete Freund's adjuvant, Example 5 above AIA was induced by intra-articular injection of methylated BSA into the right knee on day 0 as described in For the study of cgtx-544 peptide efficacy and sustained therapy effect, rats (10 subjects) were given a single intravenous injection of 1 mg/kg cgtx-544 peptide on day 1 (d1) after arthritis induction. On day 4 (d4), the cgtx-544 regimen group was divided into two subgroups, each with 5 subjects assigned. The first subgroup received a second injection of cgtx-544 to assess whether the second injection would cause a further reduction in swelling. No additional doses were administered to the second subgroup. Control animals received a single injection of vehicle (0.9% NaCl) on day 1.

Arthritis severity was monitored by measuring the knee joint diameter in triplicate using a caliper. Values were normalized to Day-21. The difference in knee swelling values was calculated by subtracting the left (non-induced) knee diameter from the right (induced) knee diameter.

2. Results

A single injection (1 x d1) with cgtx-544 (1 mg/kg BW) resulted in a clear and statistically significant reduction in knee swelling in treated animals 24 hours after application ( FIG. 25 ). Swelling could be reduced back to a significant value on day 4 after the second single injection (1 x d1 and 1 x d4). Knee swelling remained at the same level after the first and second injections and did not increase.

For statistical analysis of the results, Welsh's two-sample t-test was used. Next, statistical analyzes of both treatment regimens (1 x d1 or 1 x d1 and 1 x d4) of cgtx-544 are presented for days 2 (before dividing groups) and 5 (divided groups). Both treatment regimens showed a statistically significant reduction in knee swelling 24 hours after each injection when compared to the vehicle control group ( FIG. 26 ).

In summary, cgtx-544 peptide therapy in the AIA model showed a strong and lasting effect on inflammatory arthritis symptoms, particularly knee swelling, with swelling reduction onset 24 hours after a single injection. Even 3 days after a single injection, the cgtx-544 peptide clearly showed sustained activity and clinical effect, while the level of knee swelling remained constant. Thus, the peptide does not need to be administered daily for a sustained therapeutic effect in the AIA rat model. Therefore, i.v. In the case of dosing, it may be possible to achieve a treatment interval of 3-4 days (or longer in the case of potential s.c. routes of administration).

Example 6 - AIA rat in the model methotrexate (MTX) therapy and cgtx Comparison of -544 peptide (SEQ ID NO: 25) regimens

One. AIA rat in the model methotrexate (MTX) therapy and cgtx Comparison of -544 (SEQ ID NO: 25) regimens

Depending on the type and severity of the disease, therapy with methotrexate (MTX) differs in its dosage and regimen. On a par with standard of care, we administered 1 mg/kg body weight 1 x weekly s.c. in the AIA rat model. High-dose MTX regimen administered (Group J, n=7) and 100 μg/kg body weight 1× daily i.v. A low-dose MTX regimen administered (group L, n=7) was examined. The high-dose MTX regimen was started on the first day of the experiment (Day-21), with three additional doses (Day-14, Day -7, Day 0). The low-dose MTX regimen was started 3 days before arthritis induction (day -3) and lasted an additional 7 days after arthritis induction. The results were reported for cgtx-544 therapy group I (1 mg/kg body weight cgtx-544, same regimen as group L) and vehicle control H (0.9% NaCl administered on the day of induction, i.e., 1 x daily i.v. ) and compared with Arthritis severity was monitored by measuring the knee joint diameter in triplicate using a caliper. EDTA leukocyte samples were taken regularly to monitor immune status.

1.1 MTX therapy reduces knee swelling to a lesser extent compared to cgtx-544 therapy

First, knee parameters of the high-dose (J) and low-dose (L) MTX regimen groups are compared to the vehicle control group (H), suggesting possible differences in efficacy depending on dose and regimen. Second, 3 groups using the same regimen (applied daily i.v.) are compared: low dose (L) MTX therapy group, cgtx-544 therapy group (I) and vehicle control group (H). The difference in absolute increase in swelling was calculated by subtracting the left (non-induced) knee diameter from the right (induced) knee diameter. The calculated difference at the start of the experiment in both the MTX high-dose regimen and the low-dose regimen group was close to zero (0.01 ± 0.06 mm and 0.0 ± 0.03 mm, respectively), because both knees were nearly equal in size. . At 1 day after arthritis induction, the difference rises to 3.17 ± 1.26 mm and 3.45 ± 0.87 mm, respectively ( FIG. 11 ).

In contrast to the cgtx-544 therapy group, the reduction in swelling is not very rapid when compared to the vehicle control group with an absolute difference of 3.79±1.10 mm ( FIG. 12 ).

When values were normalized using a scale between 0 and 1, the relative increase in swelling was set to 0 at the start of the experiment (day-21), and the difference in the relative increase in swelling was calculated as before. The relative differences between the MTX high-dose regimen group and the low-dose regimen group were 0.39 ± 0.16 and 0.43 ± 0.11, respectively ( FIG. 13 ).

In comparison, the vehicle control group and the cgtx-544 therapy group had 0.46±0.14 and 0.18±0.05, respectively ( FIG. 14 ). To summarize, compared to vehicle control, we observed a mean reduction in knee swelling of 15% in MTX high-dose regimen rats and a mean 7% reduction in knee swelling in MTX low-dose regimen rats, 24 hours after arthritis induction. compared to a 60% reduction in cgtx-544-treated rats.

1.2 Cell proliferation inhibitory effect of MTX on proliferative cells

To screen for the health status of all subjects, the complete haemogram was used again. EDTA leukocytes from 7 subjects from both the MTX high-dose regimen group and the low-dose regimen group were collected weekly prior to induction, on the day of induction and on days 4 and 7 post-induction (Days-21, 14, -7). On days, 0, 4, and 7; when i.v. application was initiated, the MTX low-dose regimen group was additionally administered on days -3), measured with a Sysmex XT-2000i.V. Hematological data from untreated animals (day-21) are on par with the data provided in the literature above.

1.2.1 White Blood Cells

At the start of the experiment (Day-21), the leukocyte counts in the MTX high-dose regimen group and the low-dose regimen group were 9.41 ± 1.42 x 10 ³ /μl and 11.66 ± 2.81 x 10 ³ /μl, respectively. Due to the inflammatory response of the immune system, WBC counts rose to 20.79 ± 5.96 x 10 ³ /μl and 24.01 ± 3.81 x 10 ³ /μl, respectively, after the first and second immunizations (day -7). The values are nearly equivalent to the vehicle control group and the cgtx-544 therapy group. Within 7 days, WBC counts decreased slightly. At 4 days after arthritis induction, the WBC counts of the MTX high-dose regimen increased again, presumably due to the acute inflammatory process associated with the induction. It cannot be said whether this effect is also present in the cgtx-544 therapy group and the vehicle control group, since EDTA leukocytes were not analyzed on this date for these two groups. In addition, for the MTX low-dose regimen group, WBC count values cannot be provided as a suitable comparison, since the cytotoxic effect of MTX may affect blood composition 4 days after arthritis induction. After an additional 7 days of arthritis induction, the WBC counts were further reduced to 14.68 ± 2.15 x 10 ³ /μl and 6.63 ± 1.37 x 10 ³ /μl in both the MTX high-dose and low-dose groups, respectively. (Day 7) ( FIG. 15 ), which shows the cytostatic effect of low-dose MTX therapy. The WBC count was reduced by almost 50% compared to the start of the experiment. In contrast, the WBC counts of the MTX high-dose regimen group are nearly equivalent to those of the cgtx-544 regimen group and the vehicle control group.

1.2.2 Neutrophil Granulocytes

At the start of the experiment (day-21), the absolute neutrophil granulocyte counts of the MTX high-dose regimen and low-dose regimen were 1.55 ± 0.52 x 10 ³ /μl and 1.93 ± 0.6 x 10 ³ /μl, respectively (Fig. 16A). ) [equivalent to 16.39 ± 4.85% relative neutrophils of total WBC counts and 16.64 ± 4.07% relative neutrophils of total WBC counts, respectively ( FIG. 16B )]. As a WBC count, the neutrophil granulocyte count also rose to 7.54 ± 3.19 x 10 ³ /μl and 10.77 ± 2.7 x 10 ³ /μl, respectively, after the first and second immunizations (day -7, 35.46 ± 7.08 of WBC, respectively) % and equivalent to 44.33 ± 5.4% of WBC). Within 7 days, the count of neutrophil granulocytes decreased, and the count of the MTX high-dose therapy group at 7 days after induction was almost equivalent to the count at the start of the experiment (1.94 ± 1.1 x 10 ³ /μl; 12.63 ± 6.1% of WBC). equivalent to). This effect was also observed in the cgtx-544 therapy group and vehicle control group. The MTX low dose regimen group showed a much higher reduction, to 0.33 ± 0.36 x 10 ³ /μl neutrophil granulocytes (day 7; equivalent to 4.41 ± 4.63% of WBC).

1.2.3 Lymphocytes

At the start of the experiment (day-21), the absolute lymphocyte counts of the MTX high-dose regimen group and the low-dose regimen group were 7.39 ± 1.05 x 10 ³ /μl and 9.21 ± 2.4 x 10 ³ /μl, respectively (Fig. 17a). [equivalent to 78.77 ± 4.76% of WBC and 78.97 ± 3.94% of WBC, respectively ( FIG. 17B )]. Lymphocyte counts in both the MTX high-dose regimen group and the low-dose regimen group did not rise significantly after the first and second immunizations, differently from WBC and neutrophil granulocyte counts, respectively, 11.68 ± 2.8 x 10 ³ /μl and 11.83 counted as ± 1.35 x 10 ³ /μl (day -7). This is equivalent to 57.01 ± 6.45% of WBC and 49.83 ± 5.45% of WBC, respectively (Day -7) and represents a decreased percentage, due to a strong increase in total WBC counts. Within 7 days, the percentage of lymphocytes in WBC increased again, and in the MTX high-dose regimen group, 7 days after induction, it was almost equal to the percentage at the start of the study (78.16 ± 7.20% of WBC; day 7), which was much different from the higher (93.13 ± 4.66% of WBC; Day 7) MTX low-dose regimen group. When compared to the start of the experiment, the absolute counts of the MTX high-dose regimen increased and the absolute counts of the MTX low-dose regimen decreased: 11.38 ± 1.15 x 10 ³ /μl and 6.14 ± 1.11 x 10 ³ /μl, respectively. 7 days). Although the absolute lymphocyte count in the MTX low-dose regimen group decreased 7 days after arthritis induction compared to the start of the experiment, it still represents about 93% of all leukocytes. Thus, it can be assumed that other subpopulations of leukocytes are more affected by the cytotoxic effect than the lymphocyte population.

Treatment with MTX inhibits dihydrofolate reductase and results in a cytostatic effect on proliferating cells. Unlike subjects on cgtx-544 therapy, which did not show systemic immunosuppression, MTX-treated subjects showed different effects on hemograms when compared to vehicle controls. Seven days after arthritis induction, low-dose treated subjects had decreased WBC counts, neutrophil granulocyte counts and lymphocyte counts. Moreover, this resulted in a different composition for all leukocyte percentages. Moreover, we observed a much smaller reduction in knee swelling (7% and 15%) with both MTX regimens compared to 60% in cgtx-544-treated rats 24 hours after arthritis induction.

For statistical analysis of MTX efficacy results, Welsh's two-sample t-test was used. Next, statistical analyzes of all MTX dose levels tested (0.1 mg/kg BW i.v. and 1 mg/kg BW sc.) are presented for days 1 and 3 ( FIG. 28 ).

In summary, treatment with MTX inhibits dihydrofolate reductase and results in a cytostatic effect on proliferating cells. Unlike subjects on cgtx-544 peptide therapy, MTX-treated subjects showed different effects on hemograms when compared to vehicle controls. Seven days after arthritis induction, low-dose treated subjects had decreased WBC counts, neutrophil granulocyte counts and lymphocyte counts. Additionally, this resulted in a different composition for all white blood cell percentages. Moreover, we observed a much smaller reduction in knee swelling (7% and 15%) with both MTX regimens compared to up to 70% in cgtx-544 peptide therapy rats 24 hours after arthritis induction.

Example 7 - cgtx Confirmation of additional properties of -544

The cgtx-544 peptide consists of 38 natural amino acids without modification and has a molecular weight of approximately 4220 Da. It shares certain functional domain similarities with the α-KTx6 subfamily of scorpion peptides. cgtx-544 is controlled by four disulfide bridges, and its 3D structure contains an α-helix/β-sheet conformation.

cgtx Preparation of -544

29 provides a schematic review of the steps in the preparation of the cgtx-544 peptide. Peptide synthesis is performed on insoluble polystyrol resin using the F-moc strategy. The free N-terminal amine of the solid phase attached peptide is coupled with a single N-protected amino acid unit. Deprotection of this unit then (cleaving the F-moc group by piperidine at basic pH) gives rise to a new N-terminal amine to which additional amino acids can be attached. The Tert-butyloxycarbonyl (Boc) group serves as a permanent side chain protecting group, which is cleaved by treatment with TFA (trifluoroacetic acid) after completion of peptide synthesis.

After completion of peptide synthesis, this peptide was deprotected and cleaved from polystyrol resin. At this time point, the crude peptide product displayed a purity of about 50%, followed by analytical UPLC and LC-MS. In a subsequent step, purification of the crude peptide by preparative HPLC yields a linear peptide product with a purity of >90%.

cgtx of -544 peptides folding

These peptide solutions are folded by oxidation (formation of disulfide bonds). The folding method is based on bond formation in a phosphate buffer at pH 8.3 in the presence of atmospheric oxygen. HPLC analysis of folded cgtx-544 according to this protocol showed that the peptide was present as a mixture of folded and partially folded variants. This intermediate folding product is referred to as cgtx-544 (Mix). Based on electrophysiological data generated using different isolated UPLC-pits of material, one peak in the folding mixture (retention time 12.77 min) correlated with the natively active peptide, while all other peaks were electrophysiological. It was concluded that there was no correlation with the biological activity (FIG. 30). Thus, after the folding reaction, the active peptide (retention time 12.77 min) was purified by preparative HPLC from a cgtx-544 mixture of folded and partially folded peptides. After this purification step, the active peptide - referred to as cgtx-544 (Sing) - was typically estimated to have a purity greater than 95%. The purified cgtx-544(Sing) peptide is highly soluble and stable in 0.9% NaCl solution for several weeks at RT and 4°C.

Due to process development, the purified peptide cgtx-544 (Sing) was produced only in a small-scale format. Therefore, the material was limited and it was used in the experiment intended to confirm the IC ₅₀ value of cgtx-544(Sing) for Kv1.3, Kv1.1, Kv1.2 and Kv1.5 (see Example 8). . The crude folding mixture of cgtx-544 [cgtx-544(Mix)] was used in all other experiments described herein unless otherwise indicated by the term cgtx-544 (sing) (Examples 1 to 6). The content of active substance in this mixture was calculated to be 15%.

In summary, the cgtx-544 peptide exhibits the following biochemical properties that are important for biochemical analysis:

• cgtx-544 is a highly basic peptide containing 6 lysine and 2 arginine amino acids and can attach to the surface of glass vials at low concentrations (<100 ng/ml).

• Contains 8 cysteine amino acids, which are linked by 4 disulfide bridges.

● Isoelectric point: 9.29.

● MW when disulfide bridge is bonded: 4212 Da; MW in reduced form: 4220.2 Da.

● Because there are 4 disulfide bridges, the structure of cgtx-544 is compact.

Example 8 - based on electrophysiological analysis cgtx Confirmation of additional characterization of ion channel selectivity of -544 (sing)

1. Materials and Methods

The ion channels were baculovirus (Kv1.1-, Kv1.3- and Kv1.5-constructs) infected Sf21 cells or stably transfected CHO cells (Kv1.2) as described in Examples 1, 2, and 4 was expressed through Electrophysiological analyzes were performed on an automated patch clamp device "Patchliner" (Nanion Technology; Munich, Germany). This patchliner is a borosilicate glass chip (NPC-16) instead of a glass pipette, based on the planar patch clamp method. The patchliner can record 4 cells simultaneously. All channel recordings were made in a whole-cell manner. Data recording was performed using patchmaster software [HEKA Electronics (Lambrecht/Paltz, Germany)]. Visualization of experimental results was realized using IGOR software (Wavemetrics, USA). Mean data are presented as mean ± standard error of the mean (SEM).

Test compounds were used as concentrated stock solutions (mainly 1.5 μg/μl). The stock solution was diluted in buffer to the required final concentration.

2. Results

2.1 IC ₅₀ - target channel Kv1 .3 analysis

After chemical analysis (see Example 7), the peptide was folded by oxidation with atmospheric oxygen, and this correctly folded (biologically active) peptide fraction was purified by preparative HPLC. This purified cgtx-544 peptide was designated cgtx-544 (Sing) for a single peak.

Electrophysiological analysis of cgtx-544(Sing) resulted in an excellent IC ₅₀ value of 6.9 nM ( FIG. 31 ).

2.2 Kv1.1, Kv1 .2 and Kv1 .5 on cgtx Electrophysiological analysis of -544(Sing)

Electrophysiological analysis of cgtx-544 (Sing) on Kv1.1, Kv1.2 and Kv1.5 demonstrated that cgtx-544 was highly selective. The results of electrophysiological characterization of cgtx-544 (Sing) on Kv1.1, Kv1.2 and Kv1.5 are summarized in FIG. 32 .

Example 9 - cgtx Protease resistance of -544 (Sing)

In addition to the experiments described in Example 3, the stability of the cgtx-544 peptide was tested in blood serum samples under in vitro conditions. Therefore, the stability of cgtx-544(Sing) in human blood serum was determined by adding a known amount of cgtx-544(Sing) to human blood serum and incubating continuously at 37°C. The blocking activity of ctx-544(Sing) was measured on the Kv1.3 channel over a period of 57 days. cgtx-544(Sing) is extremely stable in human blood serum. Its activity remains constant for at least 16 hours and slowly decays until after 57 days no further peptide blocking effect can be detected. This demonstrates that cgtx-544 is highly resistant to the action of blood proteases ( FIG. 33 ).

No decrease in blocking potency was observed compared to measurements performed in the absence of serum, with IC50s in the range of 7 nM. The serum control sample without peptide showed no non-specific blocking of Kv1.3. Therefore, it was concluded that the observed blocking effect was solely due to the effect of cgtx-544(Sing). The 50% activity of cgtx-544(Sing) was preserved for 5 days in human serum at 37°C, although the concentration of cgtx-544(Sing) was measured in the solution incubated over 57 days.

In summary, the persistence of cgtx-544 in human serum at 37°C is very high. cgtx-544 does not bind (if it does) to serum components, but only with extremely low affinity) and is extremely resistant to degradation by proteases present in human serum.

Example 10 - cgtx -544 (Sing) of in vivo half-life

The purpose of this study was to implement a bioassay to evaluate the in vivo half-life of cgtx-544(Sing) in rats. Previous experiments (Example 3) have shown that cgtx-544 does not degrade rapidly when spiked into serum and incubated at 37°C.

1. Materials and Methods

Rats were given cgtx-544(Sing) (750 μg/kg BW) i.v. injected. Blood was drawn at time points 0, 1, 10 and 60 minutes. Subsequently, serum was prepared from blood samples. The cgtx-544 content in these samples was analyzed electrophysiologically by measuring the blocking effect on Kv1.3 channels expressed in CHO cells. The serum control sample without peptide (0 min) showed no non-specific blocking of Kv1.3, and the cgtx-544(Sing) activity of the 1 min sample was considered as 100%. Samples collected at the third time point (60 min) showed 10% of the original blocking activity. To study the amount of unbound cgtx-544 (Sing) in the rat, the amount of cgtx-544 (Sing) present in the circulating blood at the above-mentioned time points was calculated through a calibration curve. This calibration curve was obtained by adding a known amount of cgtx-544 (Sing) to blood serum and subsequently measuring electrophysiologically on the Kv1.3 channel. Blockade of Kv1.3 currents by these spiked samples was similar to that obtained with cgtx-544(Sing) in the absence of serum (data not shown).

2. Results

The breakdown of cgtx-544(Sing) in the blood of treated rats follows a typical decay curve, yielding a half-life of approximately t _1/2 =26 minutes ( FIG. 34 ), and cgtx-544 in circulating blood The approximate concentration of (Sing) is 62 nM. This concentration is sufficient to achieve complete blockage of the Kv1.3 channel. The half-life is within the range observed for other peptide channel blockers - variants of the ShK toxin display a half-life of 20-50 minutes.

In summary, in rats i.v. The in vivo half-life of cgtx-544 after application is within the range of the competitor peptide ShK. Its in vivo half-life is approximately 26 minutes. Because of the small size and compact structure of cgtx-544, the peptide is presumably removed from the blood through renal filtration. It is reasonable to assume that the half-life of cgtx-544 in humans is longer than in rats, since rats have a much faster metabolic rate than humans. The in vivo half-life does not correspond to the duration of the drug effect, since the peptide binds tightly to the ion channel and blocks it for a long time.

Example 11 - Clinical data

To date, no clinical studies have been conducted.

First human study

A phase 1 study may be a randomized, double-blind, placebo-controlled, single center trial in healthy subjects.

The first part of this study was a single-dose, dose-escalation study in a cohort of 4 subjects (3 + 1) for the lower dose group and a cohort of 8 subjects (2 + 6) for the higher dose group. can be The starting dose will be estimated from the NOAEL in toxicity studies as specified in the industry's FDA guidance "Predicting the Maximum Safe Starting Dose in Early Clinical Trials for Treatment of Healthy Adult Volunteers".

The second part of phase 1a can be designed as a multi-dose study. One cohort of 8 subjects (6 + 2) will be exposed to one dose level below the MTD for daily application for 7 consecutive days.

The primary goal of this standard phase 1a study may be to evaluate the safety, tolerability and pharmacokinetics of cgtx-544 in healthy subjects.

proof-of-concept study

This Phase 2a study could be a randomized, double-blind, placebo-controlled, multi-center, proof-of-concept and dose investigation study in patients with moderate to severe plaque psoriasis.

The planned phase 2a proof-of-concept study will involve 80 adult patients (20 + 60) and 3 dose levels. Each dose level will be tested in 20 patients over a period of 12 weeks.

Primary endpoint: Proportion of subjects achieving PASI 75 at week 12 (patients exhibiting at least 75% improvement from baseline PASI).

Secondary endpoints: Evaluate the safety of cgtx-544 for up to 12 weeks by evaluating adverse events, vital signs, laboratory parameters, physical examination, and EEG.

Pharmacokinetics of cgtx-544 at different time points during 12 weeks after first treatment. Proportion of subjects achieving a “clean” or “nearly clear” IGA score at week 12.

Proportion of subjects achieving PASI 50 at week 12 (patients exhibiting at least 50% improvement from baseline PASI).

Proportion of subjects achieving PASI 90 at week 12 (patients exhibiting at least 90% improvement from baseline PASI).

A similar study can be conducted for vasculitis. Endpoints will be evaluated accordingly.

SEQUENCE LISTING <110> conoGenetix biosciences GmbH <120> Kv1.3 potassium channel antagonists <130> C 9297 <160> 32 <170> PatentIn version 3.5 <210> 1 <211> 34 <212> PRT <213> Artificial Sequence < 220> <223> peptide compound <220> <221> VARIANT <222> (1)..(1) <223> /replace="Val" /replace="Ile" /replace="Leu" <220> < 221> VARIANT <222> (2)..(2) <223> /replace="Arg" /replace="Lys" /replace="Thr" /replace="Tyr" <220> <221> VARIANT <222 > (4)..(4) <223> /replace="Thr" /replace="Lys" /replace="Ser" /replace="Tyr" <220> <221> VARIANT <222> (5). .(5) <223> /replace="Gly" /replace="Ser" /replace="Asn" /replace="Ile" /replace="Lys" /replace="Gln" /replace="Ala" / replace="Val" /replace="Leu" /replace="Tyr" <220> <221> VARIANT <222> (6)..(6) <223> /replace="Ser" /replace="Thr" <220> <221> VARIANT <222> (7)..(7) <223> /replace="Arg" /replace="Pro" <220> <221> VARIANT <222> (8)..(8 ) <223> /replace="Gln" /replace="Asn" /replace="Glu" <220> <221> VARIANT <222> (10)..(10) <223> /replace="Tyr" / replace=" Ile" /replace="Leu" /replace="Trp" /replace="Ser" /replace="Thr" /replace="Val" /replace="Leu" /replace="Phe" <220> <221> VARIANT <222> (11)..(11) <223> /replace="Arg" /replace="Pro" /replace="Lys" /replace="Glu" /replace="Ser" /replace="Thr " /replace="Tyr" <220> <221> VARIANT <222> (12)..(12) <223> /replace="His" /replace="Val" /replace="Ile" /replace=" Leu" /replace="Ala" <220> <221> VARIANT <222> (14)..(14) <223> /replace="Lys" /replace="Gln" /replace="Asn" <220> <221> VARIANT <222> (15)..(15) <223> /replace="Asp" /replace="Ala" /replace="Arg" /replace="Glu" /replace="Val" /replace ="Leu" /replace="Ile" <220> <221> VARIANT <222> (16)..(16) <223> /replace="Gln" /replace="Ala" /replace="Leu" / replace="Asp" /replace="Asn" /replace="Val" /replace="Ile" <220> <221> VARIANT <222> (21)..(21) <223> /replace="Tyr" /replace="Ser" /replace="Thr" /replace="Gln" <220> <221> VARIANT <222> (22)..(22) <223> /replace="Gly" /replace="Val " /replace="Ile" /replace="Leu" <220> <221> VARIANT <222> (30)..(3 0) <223> /replace="Arg" <220> <221> VARIANT <222> (32)..(32) <223> /replace="Asn" /replace="Gln" /replace="Thr" /replace="Ser" <220> <221> VARIANT <222> (33)..(33) <223> /replace="Arg" /replace="Lys" <400> 1 Ala Ser Cys Arg Thr Pro Lys Asp Cys Ala Asp Pro Cys Arg Lys Glu 1 5 10 15 Thr Gly Cys Pro Asn Ala Lys Cys Met Asn Arg Lys Cys Lys Cys Tyr 20 25 30 Gly Cys <210> 2 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220> <221> VARIANT <222> (1)..(1) <223> /replace="Val" /replace="Ile" <220> <221> VARIANT < 222> (2)..(2) <223> /replace="Arg" /replace="Lys" <220> <221> VARIANT <222> (4)..(4) <223> /replace=" Thr" /replace="Lys" <220> <221> VARIANT <222> (5)..(5) <223> /replace="Gly" /replace="Ser" /replace="Asn" /replace= "Ile" <220> <221> VARIANT <222> (6)..(6) <223> /replace="Ser" <220> <221> VARIANT <222> (7)..(7) <223 > /replace="Arg" /replace="Pro" <220> <221> VARIANT <222> (8)..(8) <223> /replace="Gln" /replace="Asn" /replace=" Glu" <220> <221> VA RIANT <222> (10)..(10) <223> /replace="Tyr" /replace="Ile" /replace="Leu" /replace="Trp" <220> <221> VARIANT <222> ( 11)..(11) <223> /replace="Arg" /replace="Pro" /replace="Lys" /replace="Glu" /replace="Ser" <220> <221> VARIANT <222> (12)..(12) <223> /replace="His" /replace="Val" <220> <221> VARIANT <222> (14)..(14) <223> /replace="Lys" /replace="Gln" <220> <221> VARIANT <222> (15)..(15) <223> /replace="Asp" /replace="Ala" /replace="Arg" <220> <221 > VARIANT <222> (16)..(16) <223> /replace="Gln" /replace="Ala" /replace="Leu" <220> <221> VARIANT <222> (21)..( 21) <223> /replace="Tyr" <220> <221> VARIANT <222> (22)..(22) <223> /replace="Gly" <220> <221> VARIANT <222> (30 )..(30) <223> /replace="Arg" <220> <221> VARIANT <222> (32)..(32) <223> /replace="Asn" <220> <221> VARIANT < 222> (33)..(33) <223> /replace="Arg" <400> 2 Ala Ser Cys Arg Thr Pro Lys Asp Cys Ala Asp Pro Cys Arg Lys Glu 1 5 10 15 Thr Gly Cys Pro Asn Ala Lys Cys Met Asn Arg Lys Cys Lys Cys Tyr 20 25 30 Gly Cys <210 > 3 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220> <221> VARIANT <222> (1)..(1) <223> /replace="Val" /replace="Ile" /replace="Leu" <220> <221> VARIANT <222> (2)..(2) <223> /replace="Arg" /replace="Lys" /replace="Thr " /replace="Tyr" <220> <221> VARIANT <222> (4)..(4) <223> /replace="Thr" /replace="Lys" /replace="Ser" /replace=" Tyr" <220> <221> VARIANT <222> (5)..(5) <223> /replace="Gly" /replace="Ser" /replace="Asn" /replace="Ile" /replace= "Lys" /replace="Gln" /replace="Ala" /replace="Val" /replace="Leu" /replace="Tyr" <220> <221> VARIANT <222> (6)..(6 ) <223> /replace="Ser" /replace="Thr" <220> <221> VARIANT <222> (7)..(7) <223> /replace="Arg" /replace="Pro" < 220> <221> VARIANT <222> (8)..(8) <223> /replace="Gln" /replace="Asn" /replace="Glu" <220> <221> VARIANT <222> (10 )..(10) <223> /replace="Tyr" /replace="Ile" /replace="Leu" /replace="Trp" /replace="Ser" /replace="Thr" /replace="Val " /replace="Leu" /replace="Phe" <220> <221> VARIANT <222> (11)..(11) <223> /replace="Arg" /replace="Pro" /replace="Lys" /replace="Glu" /replace="Ser" /replace="Thr" /replace="Tyr" <220> <221 > VARIANT <222> (12)..(12) <223> /replace="His" /replace="Val" /replace="Ile" /replace="Leu" /replace="Ala" <220> < 221> VARIANT <222> (14)..(14) <223> /replace="Lys" /replace="Gln" /replace="Asn" <220> <221> VARIANT <222> (15).. (15) <223> /replace="Asp" /replace="Ala" /replace="Arg" /replace="Glu" /replace="Val" /replace="Leu" /replace="Ile" <220 > <221> VARIANT <222> (16)..(16) <223> /replace="Gln" /replace="Ala" /replace="Leu" /replace="Asp" /replace="Asn" / replace="Val" /replace="Ile" <220> <221> VARIANT <222> (30)..(30) <223> /replace="Arg" <220> <221> VARIANT <222> (32 )..(32) <223> /replace="Asn" /replace="Gln" /replace="Thr" /replace="Ser" <220> <221> VARIANT <222> (33)..(33 ) <223> /replace="Arg" /replace="Lys" <400> 3 Ala Ser Cys Arg Thr Pro Lys Asp Cys Ala Asp Pro Cys Arg Lys Glu 1 5 10 15 Thr Gly Cys Pro Asn Ala Lys Cys Met Asn Arg Lys Cys Lys C ys Tyr 20 25 30 Gly Cys <210> 4 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220> <221> VARIANT <222> (1)..(1) <223> /replace="Val" /replace="Ile" <220> <221> VARIANT <222> (2)..(2) <223> /replace="Arg" /replace="Lys" <220 > <221> VARIANT <222> (4)..(4) <223> /replace="Thr" /replace="Lys" <220> <221> VARIANT <222> (5)..(5) < 223> /replace="Gly" /replace="Ser" /replace="Asn" /replace="Ile" <220> <221> VARIANT <222> (6)..(6) <223> /replace= "Ser" <220> <221> VARIANT <222> (7)..(7) <223> /replace="Arg" /replace="Pro" <220> <221> VARIANT <222> (8). .(8) <223> /replace="Gln" /replace="Asn" /replace="Glu" <220> <221> VARIANT <222> (10)..(10) <223> /replace=" Tyr" /replace="Ile" /replace="Leu" /replace="Trp" <220> <221> VARIANT <222> (11)..(11) <223> /replace="Arg" /replace= "Pro" /replace="Lys" /replace="Glu" /replace="Ser" <220> <221> VARIANT <222> (12)..(12) <223> /replace="His" /replace ="Val" <220> <221> VARIANT <222> (14)..(14) <223> /replace="Lys" /replace="Gln" <220> <221> VARIANT <222> (15)..(15) <223> /replace="Asp" /replace="Ala" /replace="Arg" <220> <221> VARIANT <222> ( 16)..(16) <223> /replace="Gln" /replace="Ala" /replace="Leu" <220> <221> VARIANT <222> (30)..(30) <223> / replace="Arg" <220> <221> VARIANT <222> (32)..(32) <223> /replace="Asn" <220> <221> VARIANT <222> (33)..(33) <223> /replace="Arg" <400> 4 Ala Ser Cys Arg Thr Pro Lys Asp Cys Ala Asp Pro Cys Arg Lys Glu 1 5 10 15 Thr Gly Cys Pro Asn Ala Lys Cys Met Asn Arg Lys Cys Lys Cys Tyr 20 25 30 Gly Cys <210> 5 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220> <221> VARIANT <222> (1)..(1) <223> /replace="Val" /replace="Ile" /replace="Leu" <220> <221> VARIANT <222> (2)..(2) <223> /replace="Arg" /replace="Lys " /replace="Thr" /replace="Tyr" <220> <221> VARIANT <222> (4)..(4) <223> /replace="Thr" /replace="Lys" /replace=" Ser" /replace="Tyr" <220> <221> VARIANT <222> (5)..(5) <223> /replace="Gly" /replace="Ser" /replace="Asn" /replace= "Ile" /r eplace="Lys" /replace="Gln" /replace="Ala" /replace="Val" /replace="Leu" /replace="Tyr" <220> <221> VARIANT <222> (6).. (6) <223> /replace="Ser" /replace="Thr" <220> <221> VARIANT <222> (7)..(7) <223> /replace="Arg" /replace="Pro " <220> <221> VARIANT <222> (8)..(8) <223> /replace="Gln" /replace="Asn" /replace="Glu" <220> <221> VARIANT <222> (10)..(10) <223> /replace="Tyr" /replace="Ile" /replace="Leu" /replace="Trp" /replace="Ser" /replace="Thr" /replace= "Val" /replace="Leu" /replace="Phe" <220> <221> VARIANT <222> (11)..(11) <223> /replace="Arg" /replace="Pro" /replace ="Lys" /replace="Glu" /replace="Ser" /replace="Thr" /replace="Tyr" <220> <221> VARIANT <222> (12)..(12) <223> / replace="His" /replace="Val" /replace="Ile" /replace="Leu" /replace="Ala" <220> <221> VARIANT <222> (14)..(14) <223> /replace="Lys" /replace="Gln" /replace="Asn" <220> <221> VARIANT <222> (15)..(15) <223> /replace="Asp" /replace="Ala " /replace="Arg" /replace="Glu" /replace="Val" /replace="Leu" /replace="Ile" <220 > <221> VARIANT <222> (16)..(16) <223> /replace="Gln" /replace="Ala" /replace="Leu" /replace="Asp" /replace="Asn" / replace="Val" /replace="Ile" <220> <221> VARIANT <222> (21)..(21) <223> /replace="Tyr" /replace="Ser" /replace="Thr" /replace="Gln" <220> <221> VARIANT <222> (22)..(22) <223> /replace="Gly" /replace="Val" /replace="Ile" /replace="Leu " <220> <221> VARIANT <222> (30)..(30) <223> /replace="Arg" <400> 5 Ala Ser Cys Arg Thr Pro Lys Asp Cys Ala Asp Pro Cys Arg Lys Glu 1 5 10 15 Thr Gly Cys Pro Asn Ala Lys Cys Met Asn Arg Lys Cys Lys Cys Tyr 20 25 30 Gly Cys <210> 6 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220 > <221> VARIANT <222> (1)..(1) <223> /replace="Val" /replace="Ile" <220> <221> VARIANT <222> (2)..(2) < 223> /replace="Arg" /replace="Lys" <220> <221> VARIANT <222> (4)..(4) <223> /replace="Thr" /replace="Lys" <220> <221> VARIANT <222> (5)..(5) <223> /replace="Gly" /replace="Ser" /replace="Asn" /replace="Ile" <220> <221> VARIANT <222> (6)..(6) <223> /replace="Ser" <220> <221> VARIANT <222> (7)..(7) <223> /replace="Arg" /replace= "Pro" <220> <221> VARIANT <222> (8)..(8) <223> /replace="Gln" /replace="Asn" /replace="Glu" <220> <221> VARIANT < 222> (10)..(10) <223> /replace="Tyr" /replace="Ile" /replace="Leu" /replace="Trp" <220> <221> VARIANT <222> (11) ..(11) <223> /replace="Arg" /replace="Pro" /replace="Lys" /replace="Glu" /replace="Ser" <220> <221> VARIANT <222> (12 )..(12) <223> /replace="His" /replace="Val" <220> <221> VARIANT <222> (14)..(14) <223> /replace="Lys" /replace ="Gln" <220> <221> VARIANT <222> (15)..(15) <223> /replace="Asp" /replace="Ala" /replace="Arg" <220> <221> VARIANT <222> (16)..(16) <223> /replace="Gln" /replace="Ala" /replace="Leu" <220> <221> VARIANT <222> (21)..(21) <223> /replace="Tyr" <220> <221> VARIANT <222> (22)..(22) <223> /replace="Gly" <220> <221> VARIANT <222> (30). .(30) <223> /replace="Arg" <400> 6 Ala Ser Cys Arg Thr Pro Lys Asp Cys Ala Asp Pro Cys Arg Lys Glu 1 5 10 15 Thr Gly Cys Pro Asn Ala Lys Cys Met Asn Arg Lys Cys Lys Cys Tyr 20 25 30 Gly Cys <210> 7 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220 > <221> VARIANT <222> (1)..(1) <223> /replace="Val" /replace="Ile" /replace="Leu" <220> <221> VARIANT <222> (2) ..(2) <223> /replace="Arg" /replace="Lys" /replace="Thr" /replace="Tyr" <220> <221> VARIANT <222> (4)..(4) <223> /replace="Thr" /replace="Lys" /replace="Ser" /replace="Tyr" <220> <221> VARIANT <222> (5)..(5) <223> /replace ="Gly" /replace="Ser" /replace="Asn" /replace="Ile" /replace="Lys" /replace="Gln" /replace="Ala" /replace="Val" /replace=" Leu" /replace="Tyr" <220> <221> VARIANT <222> (6)..(6) <223> /replace="Ser" /replace="Thr" <220> <221> VARIANT <222 > (7)..(7) <223> /replace="Arg" /replace="Pro" <220> <221> VARIANT <222> (8)..(8) <223> /replace="Gln " /replace="Asn" /replace="Glu" <220> <221> VARIANT <222> (10)..(10) <223> /replace="Tyr" /replace="Ile" /replace=" Leu" /replace="Trp" /replace="Ser" /repl ace="Thr" /replace="Val" /replace="Leu" /replace="Phe" <220> <221> VARIANT <222> (11)..(11) <223> /replace="Arg" /replace="Pro" /replace="Lys" /replace="Glu" /replace="Ser" /replace="Thr" /replace="Tyr" <220> <221> VARIANT <222> (12). .(12) <223> /replace="His" /replace="Val" /replace="Ile" /replace="Leu" /replace="Ala" <220> <221> VARIANT <222> (14) ..(14) <223> /replace="Lys" /replace="Gln" /replace="Asn" <220> <221> VARIANT <222> (15)..(15) <223> /replace= "Asp" /replace="Ala" /replace="Arg" /replace="Glu" /replace="Val" /replace="Leu" /replace="Ile" <220> <221> VARIANT <222> ( 16)..(16) <223> /replace="Gln" /replace="Ala" /replace="Leu" /replace="Asp" /replace="Asn" /replace="Val" /replace=" Ile" <220> <221> VARIANT <222> (30)..(30) <223> /replace="Arg" <400> 7 Ala Ser Cys Arg Thr Pro Lys Asp Cys Ala Asp Pro Cys Arg Lys Glu 1 5 10 15 Thr Gly Cys Pro Asn Ala Lys Cys Met Asn Arg Lys Cys Lys Cys Tyr 20 25 30 Gly Cys <210> 8 <211> 34 <212> PRT <213> Artificial Sequence <220> < 223> peptide compound <220> <221> VARIANT <222> (1)..(1) <223> /replace="Val" /replace="Ile" <220> <221> VARIANT <222> (2) ..(2) <223> /replace="Arg" /replace="Lys" <220> <221> VARIANT <222> (4)..(4) <223> /replace="Thr" /replace= "Lys" <220> <221> VARIANT <222> (5)..(5) <223> /replace="Gly" /replace="Ser" /replace="Asn" /replace="Ile" <220 > <221> VARIANT <222> (6)..(6) <223> /replace="Ser" <220> <221> VARIANT <222> (7)..(7) <223> /replace=" Arg" /replace="Pro" <220> <221> VARIANT <222> (8)..(8) <223> /replace="Gln" /replace="Asn" /replace="Glu" <220> <221> VARIANT <222> (10)..(10) <223> /replace="Tyr" /replace="Ile" /replace="Leu" /replace="Trp" <220> <221> VARIANT < 222> (11)..(11) <223> /replace="Arg" /replace="Pro" /replace="Lys" /replace="Glu" /replace="Ser" <220> <221> VARIANT <222> (12)..(12) <223> /replace="His" /replace="Val" <220> <221> VARIANT <222> (14)..(14) <223> /replace= "Lys" /replace="Gln" <220> <221> VARIANT <222> (15)..(15) <223> /replace="Asp" /replace="Ala" /replace="A rg" <220> <221> VARIANT <222> (16)..(16) <223> /replace="Gln" /replace="Ala" /replace="Leu" <220> <221> VARIANT <222 > (30)..(30) <223> /replace="Arg" <400> 8 Ala Ser Cys Arg Thr Pro Lys Asp Cys Ala Asp Pro Cys Arg Lys Glu 1 5 10 15 Thr Gly Cys Pro Asn Ala Lys Cys Met Asn Arg Lys Cys Lys Cys Tyr 20 25 30 Gly Cys <210> 9 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound cgtx 538 <400> 9 Ala Ser Cys Arg Thr Pro Lys Asp Cys Ala Asp Pro Cys Arg Lys Glu 1 5 10 15 Thr Gly Cys Pro Asn Ala Lys Cys Met Asn Arg Lys Cys Lys Cys Tyr 20 25 30 Gly Cys <210> 10 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220> <221> VARIANT <222> (1)..(1) <223> /replace="Val" /replace="Ile" /replace="Leu" < 220> <221> VARIANT <222> (2)..(2) <223> /replace="Arg" /replace="Lys" /replace="Thr" /replace="Tyr" <220> <221> VARIANT <222> (4)..(4) <223> /replace="Thr" /replace="Lys" /replace="Ser" /replace="Tyr" <220> <221> VARIANT <222> ( 5)..(5) <223> /replace="Gly" /replace="Ser" /replace="Asn" /replace="Ile" /replace="Lys" /replace="Gln" /replace="Ala" /replace="Val " /replace="Leu" /replace="Tyr" <220> <221> VARIANT <222> (6)..(6) <223> /replace="Ser" /replace="Thr" <220> < 221> VARIANT <222> (7)..(7) <223> /replace="Arg" /replace="Pro" <220> <221> VARIANT <222> (10)..(10) <223> /replace="Tyr" /replace="Ile" /replace="Leu" /replace="Trp" /replace="Ser" /replace="Thr" /replace="Val" /replace="Leu" /replace ="Phe" <220> <221> VARIANT <222> (12)..(12) <223> /replace="His" /replace="Val" /replace="Ile" /replace="Leu" / replace="Ala" <220> <221> VARIANT <222> (14)..(14) <223> /replace="Lys" /replace="Gln" /replace="Asn" <220> <221> VARIANT <222> (15)..(15) <223> /replace="Asp" /replace="Ala" /replace="Arg" /replace="Glu" /replace="Val" /replace="Leu " /replace="Ile" <400> 10 Ala Ser Cys Arg Thr Pro Lys Gln Cys Ala Arg Pro Cys Arg Lys Gln 1 5 10 15 Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys Cys Lys Cys Asn 20 25 30 Arg Cys <210> 11 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220> <221> VARIANT <222> (1)..(1) <223> /replace ="Val" /replace="Ile" <220> <221> VARIANT <222> (2)..(2) <223> /replace="Arg" /replace="Lys" <220> <221> VARIANT <222> (4)..(4) <223> /replace="Thr" /replace="Lys" <220> <221> VARIANT <222> (5)..(5) <223> /replace="Gly" /replace="Ser" /replace="Asn" /replace="Ile " <220> <221> VARIANT <222> (6)..(6) <223> /replace="Ser" <220> <221> VARIANT <222> (7)..(7) <223> / replace="Arg" /replace="Pro" <220> <221> VARIANT <222> (10)..(10) <223> /replace="Tyr" /replace="Ile" /replace="Leu" /replace="Trp" <220> <221> VARIANT <222> (12)..(12) <223> /replace="His" /replace="Val" /replace="Ile" /replace="Leu " /replace="Ala" <220> <221> VARIANT <222> (14)..(14) <223> /replace="Lys" /replace="Gln" /replace="Asn" <220> < 221> VARIANT <222> (15)..(15) <223> /replace="Asp" /replace="Ala" /replace="Arg" /replace="Glu" /replace="Val" /replace= "Leu" /replace="Ile" <400> 11 Ala Ser Cys Arg Thr Pro Lys Gln Cys Ala Arg Pro Cys Arg Lys Gln 1 5 10 15 Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys Cys Lys Cys Asn 20 25 30 Arg Cys <210> 12 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound cgtx 539 <400> 12 Ala Ser Cys Arg Thr P ro Lys Gln Cys Ala Arg Pro Cys Arg Lys Gln 1 5 10 15 Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys Cys Lys Cys Asn 20 25 30 Arg Cys <210> 13 <211> 34 <212> PRT <213 > Artificial Sequence <220> <223> peptide compound <220> <221> VARIANT <222> (1)..(1) <223> /replace="Val" /replace="Ile" /replace="Leu" <220> <221> VARIANT <222> (2)..(2) <223> /replace="Arg" /replace="Lys" /replace="Thr" /replace="Tyr" <220> <221 > VARIANT <222> (5)..(5) <223> /replace="Gly" /replace="Ser" /replace="Asn" /replace="Ile" /replace="Lys" /replace=" Gln" /replace="Ala" /replace="Val" /replace="Leu" /replace="Tyr" <220> <221> VARIANT <222> (6)..(6) <223> /replace= "Ser" /replace="Thr" <220> <221> VARIANT <222> (7)..(7) <223> /replace="Arg" /replace="Pro" <220> <221> VARIANT < 222> (14)..(14) <223> /replace="Arg" /replace="Gln" /replace="Asn" <220> <221> VARIANT <222> (15)..(15) < 223> /replace="Asp" /replace="Ala" /replace="Arg" /replace="Glu" /replace="Val" /replace="Leu" /replace="Ile" <220> <221> V ARIANT <222> (16)..(16) <223> /replace="Gln" /replace="Ala" /replace="Leu" /replace="Asp" /replace="Asn" /replace="Val " /replace="Ile" <400> 13 Ala Ser Cys Arg Thr Pro Lys Gln Cys Tyr Pro His Cys Lys Lys Glu 1 5 10 15 Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys Cys Lys Cys Asn 20 25 30 Arg Cys <210> 14 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220> <221> VARIANT <222> (1)..(1) <223> /replace ="Val" /replace="Ile" <220> <221> VARIANT <222> (2)..(2) <223> /replace="Arg" /replace="Lys" <220> <221> VARIANT <222> (5)..(5) <223> /replace="Gly" /replace="Ser" /replace="Asn" /replace="Ile" <220> <221> VARIANT <222> (6 )..(6) <223> /replace="Ser" /replace="Thr" <220> <221> VARIANT <222> (7)..(7) <223> /replace="Arg" /replace ="Pro" <220> <221> VARIANT <222> (14)..(14) <223> /replace="Arg" /replace="Gln" <220> <221> VARIANT <222> (15) ..(15) <223> /replace="Asp" /replace="Ala" /replace="Arg" <220> <221> VARIANT <222> (16)..(16) <223> /replace= "Gln" /replace="Ala" /replace="Leu" <400> 14 Ala Ser Cys Arg Thr Pro Lys Gln Cys Tyr Pro His Cys Lys Lys Lys Glu 1 5 10 15 Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys Cys Lys Cys Asn 20 25 30 Arg Cys <210> 15 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound cgtx 540 <400> 15 Ala Ser Cys Arg Thr Pro Lys Gln Cys Tyr Pro His Cys Lys Lys Glu 1 5 10 15 Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys Cys Lys Cys Asn 20 25 30 Arg Cys <210> 16 <211> 34 <212> PRT <213> Artificial Sequence <220> < 223> peptide compound <220> <221> VARIANT <222> (1)..(1) <223> /replace="Val" /replace="Ile" /replace="Leu" <220> <221> VARIANT <222> (2)..(2) <223> /replace="Ser" /replace="Lys" /replace="Thr" /replace="Tyr" <220> <221> VARIANT <222> (4 )..(4) <223> /replace="Thr" /replace="Lys" /replace="Ser" /replace="Tyr" <220> <221> VARIANT <222> (5)..(5 ) <223> /replace="Gly" /replace="Ser" /replace="Asn" /replace="Ile" /replace="Lys" /replace="Gln" /replace="Ala" /repl ace="Val" /replace="Leu" /replace="Tyr" <220> <221> VARIANT <222> (6)..(6) <223> /replace="Ser" /replace="Thr" <220> <221> VARIANT <222> (7)..(7) <223> /replace="Lys" /replace="Pro" <220> <221> VARIANT <222> (8)..(8 ) <223> /replace="Gln" /replace="Asn" /replace="Glu" <220> <221> VARIANT <222> (10)..(10) <223> /replace="Tyr" / replace="Ile" /replace="Leu" /replace="Trp" /replace="Ser" /replace="Thr" /replace="Val" /replace="Leu" /replace="Phe" <220> <221> VARIANT <222> (11)..(11) <223> /replace="Arg" /replace="Pro" /replace="Lys" /replace="Glu" /replace="Ser" /replace ="Thr" /replace="Tyr" <220> <221> VARIANT <222> (12)..(12) <223> /replace="His" /replace="Val" /replace="Ile" / replace="Leu" /replace="Ala" <220> <221> VARIANT <222> (14)..(14) <223> /replace="Lys" /replace="Gln" /replace="Asn" <220> <221> VARIANT <222> (15)..(15) <223> /replace="Asp" /replace="Ala" /replace="Arg" /replace="Glu" /replace="Val " /replace="Leu" /replace="Ile" <220> <221> VARIANT <222> (16)..(16) <223> /replace= "Gln" /replace="Ala" /replace="Leu" /replace="Asp" /replace="Asn" /replace="Val" /replace="Ile" <220> <221> VARIANT <222> ( 32)..(32) <223> /replace="Tyr" /replace="Gln" /replace="Thr" /replace="Ser" <220> <221> VARIANT <222> (33)..( 33) <223> /replace="Gly" /replace="Lys" <400> 16 Ala Arg Cys Arg Thr Pro Arg Asp Cys Ala Asp Pro Cys Arg Lys Glu 1 5 10 15 Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys Cys Arg Cys Asn 20 25 30 Arg Cys <210> 17 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220> <221> VARIANT <222> (1) ..(1) <223> /replace="Val" /replace="Ile" <220> <221> VARIANT <222> (2)..(2) <223> /replace="Ser" /replace= "Lys" <220> <221> VARIANT <222> (4)..(4) <223> /replace="Thr" /replace="Lys" <220> <221> VARIANT <222> (5). .(5) <223> /replace="Gly" /replace="Ser" /replace="Asn" /replace="Ile" <220> <221> VARIANT <222> (6)..(6) < 223> /replace="Ser" /replace="Thr" <220> <221> VARIANT <222> (7)..(7) <223> /replace="Lys" /replace="Pro" <220> <221> VARIANT <222> (8)..(8) <223> /replace="Gln" /replace="Asn" /replace="Glu" <220> <221> VARIANT <222> (10)..(10 ) <223> /replace="Tyr" /replace="Ile" /replace="Leu" /replace="Trp" <220> <221> VARIANT <222> (11)..(11) <223> / replace="Arg" /replace="Pro" /replace="Lys" /replace="Glu" /replace="Ser" <220> <221> VARIANT <222> (12)..(12) <223> /replace="His" /replace="Val" <220> <221> VARIANT <222> (14)..(14) <223> /replace="Lys" /replace="Gln" <220> <221 > VARIANT <222> (15)..(15) <223> /replace="Asp" /replace="Ala" /replace="Arg" <220> <221> VARIANT <222> (16)..( 16) <223> /replace="Gln" /replace="Ala" /replace="Leu" <220> <221> VARIANT <222> (32)..(32) <223> /replace="Tyr" <220> <221> VARIANT <222> (33)..(33) <223> /replace="Gly" <400> 17 Ala Arg Cys Arg Thr Pro Arg Asp Cys Ala Asp Pro Cys Arg Lys Glu 1 5 10 15 Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys Cys Arg Cys Asn 20 25 30 Arg Cys <210> 18 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound cgtx 541 <400> 18 Ala Arg Cys Arg Thr Pro Arg Asp Cys Ala Asp Pro Cys Arg Lys Glu 1 5 10 15 Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys Cys Arg Cys Asn 20 25 30 Arg Cys <210> 19 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound cgtx 542 <400> 19 Ala Arg Cys Arg Thr Ser Arg Asn Cys Ala Lys Pro Cys Arg Lys Gln 1 5 10 15 Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys Cys Arg Cys Asn 20 25 30 Arg Cys <210> 20 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220> <221> VARIANT <222> (4)..(4) <223> /replace="Arg" /replace="Lys" /replace="Ser" /replace="Tyr" <220> <221> VARIANT <222> (5 )..(5) <223> /replace="Thr" /replace="Gly" /replace="Ser" /replace="Ile" /replace="Lys" /replace="Gln" /replace="Ala " /replace="Val" /replace="Leu" /replace="Tyr" <220> <221> VARIANT <222> (6)..(6) <223> /replace="Ser" /replace=" Thr" <220> <221> VARIANT <222> (7)..(7) <223> /replace="Arg" /replace="Pro" <220> <221> VARIANT <222> (8).. (8) <223> /replace="Asp" /replace="Asn" /replace="Glu" <220> <221> VARIANT <222> (10)..(10) <223> /replace="Ala " /replace="Ile" /replace="Leu" /replace="Trp" /replace="Ser" /replace="Thr" /replace="Val" /replace="Leu" /replace="Phe" < 220> <221> VARIANT <222> (11)..(11) <223> /replace="Asp" /replace="Arg" /replace="Lys" /replace="Glu" /replace="Ser" /replace="Thr" /replace="Tyr" <220> <221> VARIANT <222> (12)..(12) <223> /replace="Pro" /replace="Val" /replace="Ile " /replace="Leu" /replace="Ala" <220> <221> VARIANT <222> (14)..(14) <223> /replace="Arg" /replace="Gln" /replace=" Asn" <220> <221> VARIANT <222> (15)..(15) <223> /replace="Asp" /replace="Ala" /replace="Arg" /replace="Glu" /replace= "Val" /replace="Leu" /replace="Ile" <220> <221> VARIANT <222> (16)..(16) <223> /replace="Gln" /replace="Ala" /replace ="Leu" /replace="Asp" /replace="Asn" /replace="Val" /replace="Ile" <400> 20 Ile Ser Cys Thr Asn Pro Lys Gln Cys Tyr Pro His Cys Lys Lys Glu 1 5 10 15 Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys Cys Lys Cys Asn 20 25 30 Arg Cys <210> 21 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220> <221> VARIANT <222 > (4)..(4) <223> /replace="Arg" /replace="Lys" <220> <221> VARIANT <222> (5)..(5) <223> /replace="Thr " /replace="Gly" /replace="Ser" /replace="Ile" <220> <221> VARIANT <222> (6)..(6) <223> /replace="Ser" <220> < 221> VARIANT <222> (7)..(7) <223> /replace="Arg" /replace="Pro" <220> <221> VARIANT <222> (8)..(8) <223> /replace="Asp" /replace="Asn" /replace="Glu" <220> <221> VARIANT <222> (10)..(10) <223> /replace="Ala" /replace="Ile " /replace="Leu" /replace="Trp" <220> <221> VARIANT <222> (11)..(11) <223> /replace="Asp" /replace="Arg" /replace=" Lys" /replace="Glu" /replace="Ser" <220> <221> VARIANT <222> (12)..(12) <223> /replace="Pro" /replace="Val" <220> <221> VARIANT <222> (14)..(14) <223> /replace="Arg" /replace="Gln" <220> <221> VARIANT <222> (15)..(15) <223 > /replace="Asp" /replace="Ala" /replace="Arg" <220> <221> VARI ANT <222> (16)..(16) <223> /replace="Gln" /replace="Ala" /replace="Leu" <400> 21 Ile Ser Cys Thr Asn Pro Lys Gln Cys Tyr Pro His Cys Lys Lys Glu 1 5 10 15 Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys Cys Lys Cys Asn 20 25 30 Arg Cys <210> 22 <211> 34 <212> PRT <213> Artificial Sequence <220> <223 > peptide compound cgtx 545 <400> 22 Ile Ser Cys Thr Asn Pro Lys Gln Cys Tyr Pro His Cys Lys Lys Glu 1 5 10 15 Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys Cys Lys Cys Asn 20 25 30 Arg Cys <210> 23 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220> <221> VARIANT <222> (1)..(1) <223> /replace=" Gln" /replace="Ser" /replace="Tyr" /replace="Asn" <220> <221> VARIANT <222> (2)..(2) <223> /replace="Phe" /replace= "Val" /replace="Ala" /replace="Leu" /replace="Trp" <220> <221> VARIANT <222> (3)..(3) <223> /replace="Thr" /replace ="Tyr" /replace="Ser" /replace="Val" /replace="Ala" /replace="Leu" <220> <221> VARIANT <222> (6)..(6) <223> / replace ="Ser" /replace="Thr" /replace="Tyr" /replace="Arg" <220> <221> VARIANT <222> (8)..(8) <223> /replace="Arg" / replace="Lys" /replace="Ser" /replace="Tyr" <220> <221> VARIANT <222> (9)..(9) <223> /replace="Thr" /replace="Gly" /replace="Asn" /replace="Ile" /replace="Lys" /replace="Gln" /replace="Ala" /replace="Val" /replace="Leu" /replace="Tyr" <220 > <221> VARIANT <222> (10)..(10) <223> /replace="Ser" /replace="Thr" <220> <221> VARIANT <222> (11)..(11) < 223> /replace="Arg" /replace="Pro" <220> <221> VARIANT <222> (12)..(12) <223> /replace="Asp" /replace="Asn" /replace= "Glu" <220> <221> VARIANT <222> (14)..(14) <223> /replace="Ala" /replace="Tyr" /replace="Ile" /replace="Trp" /replace ="Ser" /replace="Thr" /replace="Val" /replace="Leu" /replace="Phe" <220> <221> VARIANT <222> (15)..(15) <223> / replace="Asp" /replace="Arg" /replace="Lys" /replace="Glu" /replace="Ser" /replace="Thr" /replace="Tyr" <220> <221> VARIANT <222 > (16)..(16) <223> /replace="His" /replace="Val" /replace="Ile" /replace ="Leu" /replace="Ala" <220> <221> VARIANT <222> (18)..(18) <223> /replace="Arg" /replace="Gln" /replace="Asn" < 220> <221> VARIANT <222> (19)..(19) <223> /replace="Lys" /replace="Asp" /replace="Arg" /replace="Glu" /replace="Val" /replace="Leu" /replace="Ile" <220> <221> VARIANT <222> (20)..(20) <223> /replace="Glu" /replace="Ala" /replace="Leu " /replace="Asp" /replace="Asn" /replace="Val" /replace="Ile" <220> <221> VARIANT <222> (25)..(25) <223> /replace=" Asn" /replace="Ser" /replace="Thr" /replace="Gln" <220> <221> VARIANT <222> (26)..(26) <223> /replace="Ala" /replace= "Val" /replace="Ile" /replace="Leu" <220> <221> VARIANT <222> (34)..(34) <223> /replace="Arg" <220> <221> VARIANT < 222> (36)..(36) <223> /replace="Tyr" /replace="Gln" /replace="Thr" /replace="Ser" <220> <221> VARIANT <222> (37) ..(37) <223> /replace="Gly" /replace="Lys" <400> 23 Thr Ile Ile Asn Val Lys Cys Thr Ser Pro Lys Gln Cys Leu Pro Pro 1 5 10 15 Cys Lys Ala Gln Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn A rg Lys 20 25 30 Cys Lys Cys Asn Arg Cys 35 <210> 24 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220> <221> VARIANT <222> (1) ..(1) <223> /replace="Gln" <220> <221> VARIANT <222> (2)..(2) <223> /replace="Phe" <220> <221> VARIANT <222 > (3)..(3) <223> /replace="Thr" <220> <221> VARIANT <222> (6)..(6) <223> /replace="Ser" <220> <221 > VARIANT <222> (8)..(8) <223> /replace="Arg" /replace="Lys" <220> <221> VARIANT <222> (9)..(9) <223> / replace="Thr" /replace="Gly" /replace="Asn" /replace="Ile" <220> <221> VARIANT <222> (10)..(10) <223> /replace="Ser" <220> <221> VARIANT <222> (11)..(11) <223> /replace="Arg" /replace="Pro" <220> <221> VARIANT <222> (12)..(12 ) <223> /replace="Asp" /replace="Asn" /replace="Glu" <220> <221> VARIANT <222> (14)..(14) <223> /replace="Ala" / replace="Tyr" /replace="Ile" /replace="Trp" <220> <221> VARIANT <222> (15)..(15) <223> /replace="Asp" /replace="Arg" /replace="Lys" /replace="Glu" /replace="Ser" <220> <221> VARIANT <222> (16)..(16) < 223> /replace="His" /replace="Val" <220> <221> VARIANT <222> (18)..(18) <223> /replace="Arg" /replace="Gln" <220> <221> VARIANT <222> (19)..(19) <223> /replace="Lys" /replace="Asp" /replace="Arg" <220> <221> VARIANT <222> (20). .(20) <223> /replace="Glu" /replace="Ala" /replace="Leu" <220> <221> VARIANT <222> (25)..(25) <223> /replace=" Asn" <220> <221> VARIANT <222> (26)..(26) <223> /replace="Ala" <220> <221> VARIANT <222> (34)..(34) <223> /replace="Arg" <220> <221> VARIANT <222> (36)..(36) <223> /replace="Tyr" <220> <221> VARIANT <222> (37)..(37 ) <223> /replace="Gly" <400> 24 Thr Ile Ile Asn Val Lys Cys Thr Ser Pro Lys Gln Cys Leu Pro Pro 1 5 10 15 Cys Lys Ala Gln Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys 20 25 30 Cys Lys Cys Asn Arg Cys 35 <210> 25 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> peptide compound cgtx 544 <400> 25 Thr Ile Ile Asn Val Lys Cys Thr Ser Pro Lys Gln Cys Leu Pro Pro 1 5 10 15 Cys Lys Ala Gln Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys 20 25 30 Cys Lys Cys Asn Arg Cys 35 <210> 26 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> peptide compound cgtx 547 <400> 26 Gln Phe Thr Asn Val Ser Cys Thr Thr Ser Lys Glu Cys Trp Ser Val 1 5 10 15 Cys Gln Arg Leu Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys 20 25 30 Cys Lys Cys Asn Arg Cys 35 <210> 27 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220> <221> VARIANT <222> (3)..(3) <223> /replace="Ile" /replace="Phe " /replace="Val" /replace="Ala" /replace="Leu" /replace="Trp" <220> <221> VARIANT <222> (7)..(7) <223> /replace=" Lys" /replace="Thr" /replace="Tyr" /replace="Arg" <220> <221> VARIANT <222> (9)..(9) <223> /replace="Arg" /replace= "Lys" /replace="Ser" /replace="Tyr" <220> <221> VARIANT <222> (10)..(10) <223> /replace="Thr" /replace="Ser" /replace ="Asn" /replace="Ile" /replace="Lys" /replace="Gln" /replace="Ala" /replace="Val" /replace="Leu" /replace="Tyr" <220> < 221> VARIANT <222> (11)..(11) <223> /replace="Pro" /replace="Thr" <220> <221> VARIANT <222> (12)..(12) <223> /replace="Arg" /replace="Lys" <220> <221 > VARIANT <222> (13)..(13) <223> /replace="Asp" /replace="Asn" /replace="Glu" <220> <221> VARIANT <222> (15)..( 15) <223> /replace="Ala" /replace="Tyr" /replace="Leu" /replace="Trp" /replace="Ser" /replace="Thr" /replace="Val" /replace="Leu" /replace="Phe" <220> <221> VARIANT <222> (16)..(16) <223 > /replace="Asp" /replace="Arg" /replace="Pro" /replace="Glu" /replace="Ser" /replace="Thr" /replace="Tyr" <220> <221> VARIANT <222> (17)..(17) <223> /replace="His" /replace="Val" /replace="Ile" /replace="Leu" /replace="Ala" <220> <221> VARIANT <222> (19)..(19) <223> /replace="Arg" /replace="Gln" /replace="Asn" <220> <221> VARIANT <222> (20)..(20 ) <223> /replace="Lys" /replace="Ala" /replace="Arg" /replace="Glu" /replace="Val" /replace="Leu" /replace="Ile" <220> < 221> VARIANT <222> (21)..(21) <223> /replace="Glu" /replace="Gln" /replace="Leu" /replace="Asp" /replace="Asn" /replace= "Val" /replace="Ile" <220> <221> VARIANT <222> (26)..(26) <223> /replace="Asn" /replace="Ser" /replace="Thr" /replace ="Gln" <220> <221> VARIANT <222> (27)..(27) <223> /replace="Ala" /replace="Val" /replace="Ile" /replace="Leu" < 220> <221> VARIANT <222> (35)..(35) <223> /replace="Arg" <220> <221> VARIANT <222> (37)..(37) <223> /replace="Tyr" /replace="Gln" /replace="Thr" /replace="Ser" <220> <221> VARIANT <222> ( 38)..(38) <223> /replace="Gly" /replace="Lys" <400> 27 Gly Val Pro Ile Asn Val Ser Cys Thr Gly Ser Pro Gln Cys Ile Lys 1 5 10 15 Pro Cys Lys Asp Ala Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg 20 25 30 Lys Cys Lys Cys Asn Arg Cys 35 <210> 28 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> peptide compound <220 > <221> VARIANT <222> (3)..(3) <223> /replace="Ile" /replace="Phe" <220> <221> VARIANT <222> (7)..(7) < 223> /replace="Lys" <220> <221> VARIANT <222> (9)..(9) <223> /replace="Arg" /replace="Lys" <220> <221> VARIANT <222 > (10)..(10) <223> /replace="Thr" /replace="Ser" /replace="Asn" /replace="Ile" <220> <221> VARIANT <222> (11). .(11) <223> /replace="Pro" <220> <221> VARIANT <222> (12)..(12) <223> /replace="Arg" /replace="Lys" <220> < 221> VARIANT <222> (13)..(13) <223> /replace="Asp" /replace="Asn" /replace="Glu" <220> <221> VARIANT <222> (15). .(15) <223> /replace="Ala" /replace="Tyr" /replace="Leu" /replace="Trp" <220> <221> VARIANT <222> (16)..(16) < 223> /replace="Asp" /replace="Arg" /replace="Pro" /replace="Glu" /replace="Ser" <220> <221> VARIANT <222> (17)..(17) <223> /replace="His" /replace="Val" <220> <221> VARIANT <222> (19)..(19) <223> /replace="Arg" /replace="Gln" <220 > <221> VARIANT <222> (20)..(20) <223> /replace="Lys" /replace="Ala" /replace="Arg" <220> <221> VARIANT <222> (21) ..(21) <223> /replace="Glu" /replace="Gln" /replace="Leu" <220> <221> VARIANT <222> (26)..(26) <223> /replace= "Asn" <220> <221> VARIANT <222> (27)..(27) <223> /replace="Ala" <220> <221> VARIANT <222> (35)..(35) <223 > /replace="Arg" <220> <221> VARIANT <222> (37)..(37) <223> /replace="Tyr" <220> <221> VARIANT <222> (38)..( 38) <223> /replace="Gly" <400> 28 Gly Val Pro Ile Asn Val Ser Cys Thr Gly Ser Pro Gln Cys Ile Lys 1 5 10 15 Pro Cys Lys Asp Ala Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg 20 25 30 Lys Cys Lys Cys Asn Arg Cys 35 <210> 29 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> peptide compound cgtx 543 <400> 29 Gly Val Pro Ile Asn Val Ser Cys Thr Gly Ser Pro Gln Cys Ile Lys 1 5 10 15 Pro Cys Lys Asp Ala Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg 20 25 30 Lys Cys Lys Cys Asn Arg Cys 35 <210> 30 <211> 39 <212> PRT <213> Artificial Sequence <220> < 223> peptide compound cgtx 546 <400> 30 Gly Val Ile Ile Asn Val Lys Cys Lys Ile Ser Arg Gln Cys Leu Glu 1 5 10 15 Pro Cys Lys Lys Ala Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg 20 25 30 Lys Cys Lys Cys Asn Arg Cys 35 <210> 31 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> peptide compound cgtx 548 <400> 31 Gly Val Pro Ile Asn Val Lys Cys Thr Gly Ser Pro Gln Cys Leu Lys 1 5 10 15 Pro Cys Lys Asp Ala Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg 20 25 30 Lys Cys Lys Cys Asn Arg Cys 35 <210> 32 <211> 34 <212> PRT <213> Heterometrus spinifer <400> 32 Ala Ser Cys Arg Thr Pro Lys Asp Cys Ala Asp Pro Cys A rg Lys Glu 1 5 10 15 Thr Gly Cys Pro Tyr Gly Lys Cys Met Asn Arg Lys Cys Lys Cys Asn 20 25 30Arg Cys

Claims (9)

A compound consisting of the amino acid sequence of SEQ ID NO: 25. A nucleic acid sequence encoding an amino acid sequence as recited in claim 1 . A pharmaceutical composition comprising a compound according to claim 1 for use in the treatment or prophylaxis of rheumatoid arthritis. delete delete delete delete delete delete

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